Ramon Langen

Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB.

Muscle wasting is often associated with chronic inflammation. Because tumor necrosis factor α (TNF‐α) has been implicated as a major mediator of cachexia, its effects on C2C12 myocytes were examined. TNF‐α activated nuclear factor‐κΒ (NF‐κΒ) and interfered with the expression of muscle proteins in differentiating myoblasts. Introduction of a mutant form of inhibitory protein κΒα (IκBα) restored myogenic differentiation in myoblasts treated with TNF‐α or interleukin 1β. Conversely, activation of NF‐KBby overexpression of IΚB kinase was sufficient to block myogenesis, illustrating the causal link between NF‐ΚB activation and inhibition of myogenic differentiation. The inhibitory effects of TNF‐α on myogenic differentiation were reversible, indicating that the effects of the cytokine were not due to nonspecific toxicity. Treatment of differentiated myotubes with TNF‐α did not result in a striking loss of muscle‐specific proteins, which shows that myogenesis was selectively affected in the myoblast stage by TNF‐α. An important finding was that NF‐ΚB was activated to the same extent in differentiating and differentiated cells, illustrating that once myocytes have differentiated they become refractory to the effects of NF‐ΚB activation. These results demonstrate that inflammatory cytokines may contribute to muscle wasting through the inhibition of myogenic differentiation via a NF‐κB‐dependent pathway.—Langen, R. C. J., Schols, A. M. W. J., Kelders, M. C. J. M., Wouters, E. F. M., Janssen‐Heininger, Y. M. W. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor‐KB. FASEB J. 15, 1169–1180 (2001)

Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization

Tumor necrosis factor α (TNFα) has been implicated as a mediator of muscle wasting through nuclear factor kappa B (NF‐ΚB) ‐dependent inhibition of myogenic differentiation. The aim of the present study was to identify the regulatory molecule(s) of myogenesis targeted by TNFα/NF‐κΒ signaling. TNFα interfered with cell cycle exit and repressed the accumulation of transcripts encoding muscle‐specific genes in differentiating C2C12 myoblasts. Overexpression of a p65 (RelA) mutant lacking the transcriptional activation domain attenuated the TNFα‐mediated inhibition of muscle‐specific gene transcription. The ability of muscle regulatory factor MyoD to induce muscle‐specific transcription in 10T1/2 fibroblasts was also disrupted by wild‐type p65, demonstrating that NF‐KB transcriptional activity interferes with the function of MyoD. Inhibition of muscle‐specific gene expression by TNFα was restored by overexpression of MyoD, whereas endogenous MyoD protein abundance and stability were reduced by TNFα through increased proteolysis of MyoD by the ubiquitin proteasome pathway. Last, the inhibitory effects of TNFα on myogenic differentiation were demonstrated in a mouse model of skeletal muscle regeneration, in which TNFα caused a delay in myoblast cell cycle exit. These results implicate that TNFα inhibits myogenic differentiation through destabilizing MyoD protein in a NF‐κB‐dependent manner, which interferes with skeletal muscle regeneration and may contribute to muscle wasting.—Langen, R. C. J., van der Velden, J. L. J., Schols, A. M. W. J., Kelders, M. C. J. M., Wouters, E. F. M., Janssen‐Heininger, Y. M. W. Tumor necrosis factor‐alpha inhibits myogenic differentiation through MyoD protein destabilization. FASEB J. 18, 227–237 (2004)

TNF-alpha impairs regulation of muscle oxidative phenotype: implications for cachexia?

Chronic obstructive pulmonary disease (COPD) is characterized by weight loss, muscle wasting (in advanced disease ultimately resulting in cachexia), and loss of muscle oxidative phenotype (oxphen). This study investigates the effect of inflammation (as a determinant of muscle wasting) on muscle oxphen by using cell studies combined with analyses of muscle biopsies of patients with COPD and control participants. We analyzed markers (citrate synthase, β-hydroxyacyl-CoA dehydrogenase, and cytochrome c oxidase IV) and regulators (PGC-1α, PPAR-α, and Tfam) of oxphen in vastus lateralis muscle biopsies of patients with advanced COPD and healthy smoking control participants. Here 17 of 73 patients exhibited elevated muscle TNF-α mRNA levels. In these patients, significantly lower mRNA levels of all oxidative markers/regulators were found. Interestingly, these patients also had a significantly lower body mass index and tended to have less muscle mass. In cultured muscle cells, mitochondrial protein content and myosin heavy chain isoform I (but not II) protein and mRNA levels were reduced on chronic TNF-α stimulation. TNF-α also reduced mitochondrial respiration in a nuclear factor kappaB (NF-κB) -dependent manner. Importantly, TNF-α-induced NF-κB activation decreased promoter transactivation and transcriptional activity of regulators of mitochondrial biogenesis and muscle oxphen. In conclusion, these results demonstrate that TNF-α impairs muscle oxphen in a NF-κB-dependent manner.

PPARγ inhibits NF-κB-dependent transcriptional activation in skeletal muscle

Skeletal muscle pathology associated with a chronic inflammatory disease state (e.g., skeletal muscle atrophy and insulin resistance) is a potential consequence of chronic activation of NF-kappaB. It has been demonstrated that peroxisome proliferator-activated receptors (PPARs) can exert anti-inflammatory effects by interfering with transcriptional regulation of inflammatory responses. The goal of the present study, therefore, was to evaluate whether PPAR activation affects cytokine-induced NF-kappaB activity in skeletal muscle. Using C(2)C(12) myotubes as an in vitro model of myofibers, we demonstrate that PPAR, and specifically PPARgamma, activation potently inhibits inflammatory mediator-induced NF-kappaB transcriptional activity in a time- and dose-dependent manner. Furthermore, PPARgamma activation by rosiglitazone strongly suppresses cytokine-induced transcript levels of the NF-kappaB-dependent genes intracellular adhesion molecule 1 (ICAM-1) and CXCL1 (KC), the murine homolog of IL-8, in myotubes. To verify whether muscular NF-kappaB activity in human subjects is suppressed by PPARgamma activation, we examined the effect of 8 wk of rosiglitazone treatment on muscular gene expression of ICAM-1 and IL-8 in type 2 diabetes mellitus patients. In these subjects, we observed a trend toward decreased basal expression of ICAM-1 mRNA levels. Subsequent analyses in cultured myotubes revealed that the anti-inflammatory effect of PPARgamma activation is not due to decreased RelA translocation to the nucleus or reduced RelA DNA binding. These findings demonstrate that muscle-specific inhibition of NF-kappaB activation may be an interesting therapeutic avenue for treatment of several inflammation-associated skeletal muscle abnormalities.

Interruption of Wnt Signaling Attenuates the Onset of Pressure Overload-Induced Cardiac Hypertrophy

The hypertrophic response of the heart has been recognized recently as the net result of activation of prohypertrophic and antihypertrophic pathways. Here we report the involvement of the Wnt/Frizzled pathway in the onset of cardiac hypertrophy development. Stimulation of the Wnt/Frizzled pathway activates the disheveled (Dvl) protein. Disheveled subsequently can inhibit glycogen synthase kinase-3&bgr;, a protein with potent antihypertrophic actions through diverse molecular mechanisms. In the Wnt/Frizzled pathway, inhibition of glycogen synthase kinase-3&bgr; leads to an increased amount of &bgr;-catenin, which can act as a transcription factor for several hypertrophy-associated target genes. In this study we subjected mice lacking the Dvl-1 gene and their wild-type littermates to thoracic aortic constriction for 7, 14, and 35 days. In mice lacking the Dvl-1 gene, 7 days of pressure overload-induced increases in left ventricular posterior wall thickness and expression of atrial natriuretic factor and brain natriuretic protein were attenuated compared with their wild-type littermates. &bgr;-Catenin protein amount was reduced in the group lacking the Dvl-1 gene, and an increased glycogen synthase kinase-3&bgr; activity was observed. Moreover, the increase in the amount of Ser473-phosphorylated Akt, a stimulator of cardiac hypertrophy, was lower in the group lacking the Dvl-1 gene. In conclusion, we have demonstrated that interruption of Wnt signaling in the mice lacking the Dvl-1 gene attenuates the onset of pressure overload-induced cardiac hypertrophy through mechanisms involving glycogen synthase kinase-3&bgr; and Akt. Therefore, the Wnt/Frizzled pathway may provide novel therapeutic targets for antihypertrophic therapy.

The mechanisms of cachexia underlying muscle dysfunction in COPD

Pulmonary cachexia is a prevalent, debilitating, and well-recognized feature of COPD associated with increased mortality and loss of peripheral and respiratory muscle function. The exact cause and underlying mechanisms of cachexia in COPD are still poorly understood. Increasing evidence, however, shows that pathological changes in intracellular mechanisms of muscle mass maintenance (i.e., protein turnover and myonuclear turnover) are likely involved. Potential factors triggering alterations in these mechanisms in COPD include oxidative stress, myostatin, and inflammation. In addition to muscle wasting, peripheral muscle in COPD is characterized by a fiber-type shift toward a more type II, glycolytic phenotype and an impaired oxidative capacity (collectively referred to as an impaired oxidative phenotype). Atrophied diaphragm muscle in COPD, however, displays an enhanced oxidative phenotype. Interestingly, intrinsic abnormalities in (lower limb) peripheral muscle seem more pronounced in either cachectic patients or weight loss-susceptible emphysema patients, suggesting that muscle wasting and intrinsic changes in peripheral muscles oxidative phenotype are somehow intertwined. In this manuscript, we will review alterations in mechanisms of muscle mass maintenance in COPD and discuss the involvement of oxidative stress, inflammation, and myostatin as potential triggers of cachexia. Moreover, we postulate that an impaired muscle oxidative phenotype in COPD can accelerate the process of cachexia, as it renders muscle in COPD less energy efficient, thereby contributing to an energy deficit and weight loss when not dietary compensated. Furthermore, loss of peripheral muscle oxidative phenotype may increase the muscles susceptibility to inflammation- and oxidative stress-induced muscle damage and wasting.

Regulation of mitochondrial biogenesis during myogenesis

Pathways involved in mitochondrial biogenesis associated with myogenic differentiation are poorly defined. Therefore, C(2)C(12) myoblasts were differentiated into multi-nucleated myotubes and parameters/regulators of mitochondrial biogenesis were investigated. Mitochondrial respiration, citrate synthase- and beta-hydroxyacyl-CoA dehydrogenase activity as well as protein content of complexes I, II, III and V of the mitochondrial respiratory chain increased 4-8-fold during differentiation. Additionally, an increase in the ratio of myosin heavy chain (MyHC) slow vs MyHC fast protein content was observed. PPAR transcriptional activity and transcript levels of PPAR-alpha, the PPAR co-activator PGC-1alpha, mitochondrial transcription factor A and nuclear respiratory factor 1 increased during differentiation while expression levels of PPAR-gamma decreased. In conclusion, expression and activity levels of genes known for their regulatory role in skeletal muscle oxidative capabilities parallel the increase in oxidative parameters during the myogenic program. In particular, PGC-1alpha and PPAR-alpha may be involved in the regulation of mitochondrial biogenesis during myogenesis.

Nuclear transcription factor κ B activation and protein turnover adaptations in skeletal muscle of patients with progressive stages of lung cancer cachexia

BACKGROUND Experimental models of cancer cachexia have indicated that systemic inflammation induces muscle-protein breakdown and wasting via muscular nuclear transcription factor κB (NF-κB) activation. This process may limit the efficacy of nutritional intervention. OBJECTIVES We assessed muscle NF-κB activity and protein turnover signaling in progressive stages of clinical lung cancer cachexia and assessed whether circulating factors can induce muscular NF-κB activity. DESIGN Patients with lung cancer precachexia (n = 10) and cachexia (n = 16) were cross-sectionally compared with 22 healthy control subjects. mRNA transcripts of muscle proteolytic (ubiquitin proteasome system and autophagy lysosomal pathway) and myogenic markers and protein expression of PI3K/Akt, myostatin, and autophagy signaling were measured. A multiplex analysis showed the systemic inflammatory status, whereas plasma exposure to stable NF-κB-luciferase-reporter muscle cells revealed NF-κB inducibility. RESULTS Compared with healthy control subjects, cachectic patients had reduced (appendicular) muscle mass (-10%), muscle fiber atrophy (-27%), and decreased quadriceps strength (-31%). Subtle alterations in the muscle morphology were also detectable in precachectic patients, without changes in body composition. Despite increased Akt phosphorylation, downstream phosphosubstrates glycogen synthase kinase 3β, mammalian target of rapamycin, and Forkhead box protein were unaltered. The expression of autophagy effectors B cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3 and microtubule-associated proteins 1A/1B light chain 3B gradually increased from precachectic to cachectic patients, without differences in E3 ubiquitin ligases. Systemic and local inflammation was evident in cachexia and intermediate in precachexia, but the plasma of both patients groups caused ex vivo muscle NF-κB activation. CONCLUSIONS In lung cancer, muscular NF-κB activity is induced by factors contained within the circulation. Autophagy may contribute to increased muscle proteolysis in lung cancer cachexia, whereas the absence of downstream changes in phosphosubstrates despite increased Akt phosphorylation suggests impaired anabolic signaling that may require targeted nutritional intervention.

Extrapulmonary manifestations of chronic obstructive pulmonary disease in a mouse model of chronic cigarette smoke exposure.

Cigarette smoking is the most commonly encountered risk factor for chronic obstructive pulmonary disease (COPD), reflected by irreversible airflow limitation, frequently associated with airspace enlargement and pulmonary inflammation. In addition, COPD has systemic consequences, including systemic inflammation, muscle wasting, and loss of muscle oxidative phenotype. However, the role of smoking in the development of these extrapulmonary manifestations remains rather unexplored. Mice were exposed to cigarette smoke or control air for 6 months. Subsequently, emphysema was assessed by morphometry of lung tissue, and blood cytokine and chemokine levels were determined by a multiplex assay. Soleus, plantaris, gastrocnemius, and tibialis muscles were dissected and weighed. Muscle fiber typing was performed based on I, IIA, IIB, and IIX myosin heavy-chain isoform composition. Lungs of the smoke-exposed animals showed pulmonary inflammation and emphysema. Moreover, circulating levels of primarily proinflammatory proteins, especially TNF-alpha, were elevated after smoke exposure. Despite an attenuated body weight gain, only the soleus showed a tendency toward lower muscle weight after smoke exposure. Oxidative fiber type IIA proportion was significantly reduced in the soleus. Muscle oxidative enzyme activity was slightly reduced after smoke exposure, being most prominent for citrate synthase in the soleus and tibialis. In this mouse model, chronic cigarette smoke exposure resulted in systemic features that closely resemble the early signs of the extrapulmonary manifestations observed in patients with COPD.

Fatty acid-induced NF-κB activation and insulin resistance in skeletal muscle are chain length dependent

The saturated fatty acid (SFA) palmitate induces insulin resistance in cultured skeletal muscle cells, which may be related to NF-kappaB activation. The aim of this study was to evaluate whether other SFAs also exert these effects on skeletal muscle and whether these relate to chain length. Therefore, we incubated L6 and C(2)C(12) skeletal muscle cells with four different fatty acids, caprylate (C8:0), laurate (C12:0), palmitate (C16:0), and stearate (C18:0), to study effects on GLUT4 translocation, deoxyglucose uptake, and NF-kappaB activation. Incubation of L6 cells with the long-chain FAs C16:0 and C18:0 reduced insulin-stimulated GLUT4 translocation and deoxyglucose uptake, whereas L6 cells incubated with the medium-chain FAs C8:0 and C12:0 remained insulin sensitive. Besides increasing NF-kappaB DNA binding activity in both L6 and C(2)C(12) cells, C16:0 also induced NF-kappaB transcriptional activity. C18:0 showed comparable effects, whereas the SFAs with shorter chain lengths were not able to elevate NF-kappaB transcriptional activity. Collectively, these results demonstrate that SFA-induced NF-kappaB activation coincides with insulin resistance and depends on FA chain length.