Marco Kelders

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)

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

BackgroundCigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.MethodsWe studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.ResultsWe observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.ConclusionThe observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

Loss of quadriceps muscle oxidative phenotype and decreased endurance in patients with mild-to-moderate COPD

Being well-established in advanced chronic obstructive pulmonary disease (COPD), skeletal muscle dysfunction and its underlying pathology have been scarcely investigated in patients with mild-to-moderate airflow obstruction. We hypothesized that a loss of oxidative phenotype (oxphen) associated with decreased endurance is present in the skeletal muscle of patients with mild-to-moderate COPD. In quadriceps muscle biopsies from 29 patients with COPD (forced expiratory volume in 1 s [FEV1] 58 ± 16%pred, body mass index [BMI] 26 ± 4 kg/m(2)) and 15 controls (BMI 25 ± 3 kg/m(2)) we assessed fiber type distribution, fiber cross-sectional areas (CSA), oxidative and glycolytic gene expression, OXPHOS protein levels, metabolic enzyme activity, and levels of oxidative stress markers. Quadriceps function was assessed by isokinetic dynamometry, body composition by dual-energy X-ray absorptiometry, exercise capacity by an incremental load test, and physical activity level by accelerometry. Compared with controls, patients had comparable fat-free mass index, quadriceps strength, and fiber CSA, but quadriceps endurance was decreased by 29% (P = 0.002). Patients with COPD had a clear loss of muscle oxphen: a fiber type I-to-II shift, decreased levels of OXPHOS complexes IV and V subunits (47% and 31%, respectively; P < 0.05), a decreased ratio of 3-hydroxyacyl-CoA dehydrogenase/phosphofructokinase (PFK) enzyme activities (38%, P < 0.05), and decreased peroxisome proliferator-activated receptor-γ coactivator-1α (40%; P < 0.001) vs. increased PFK (67%; P < 0.001) gene expression levels. Within the patient group, markers of oxphen were significantly positively correlated with quadriceps endurance and inversely with the increase in plasma lactate relative to work rate during the incremental test. Levels of protein carbonylation, tyrosine nitration, and malondialdehyde protein adducts were comparable between patients and controls. However, among patients, oxidative stress levels were significantly inversely correlated with markers of oxphen and quadriceps endurance. Reduced muscle endurance associated with underlying loss of muscle oxphen is already present in patients with mild-to-moderate COPD without muscle wasting.

Glycogen synthase kinase 3 suppresses myogenic differentiation through negative regulation of NFATc3.

Skeletal muscle atrophy is a prominent and disabling feature in many chronic diseases. Prevention or reversal of muscle atrophy by stimulation of skeletal muscle growth could be an important therapeutic strategy. Glycogen synthase kinase 3β (GSK-3β) has been implicated in the negative regulation of skeletal muscle growth. Since myogenic differentiation is an essential part of muscle growth, we investigated if inhibition of GSK-3β is sufficient to stimulate myogenic differentiation and whether this depended on regulation of the transcription factor nuclear factor of activated T-cells (NFAT). In both myogenically converted mouse embryonic fibroblasts and C2C12 myoblasts, deficiency of GSK-3β protein (activity) resulted in enhanced myotube formation and muscle-specific gene expression during differentiation, which was reversed by reintroduction of wild type but not kinase-inactive (K85R) GSK-3β. In addition, GSK-3β inhibition restored myogenic differentiation following calcineurin blockade, which suggested the involvement of NFAT. GSK-3β-deficient mouse embryonic fibroblasts or myoblasts displayed enhanced nuclear translocation of NFATc3 and elevated NFAT-sensitive promoter transactivation, which was reduced by reintroducing wild type, but not K85R GSK-3β. Overexpression of NFATc3 increased muscle gene promoter transactivation, which was abolished by co-expression of wild type GSK-3β. Finally, stimulation of muscle gene expression observed following GSK-3β inhibition was strongly attenuated in NFATc3-deficient myoblasts, indicating that this response requires NFATc3. Collectively, our data demonstrate negative regulation of myogenic differentiation by GSK-3β through a transcriptional mechanism that depends on NFATc3. Inhibition of GSK-3β may be a potential strategy in prevention or treatment of muscle atrophy.

ENHANCED MYOGENIC DIFFERENTIATION BY EXTRACELLULAR MATRIX IS REGULATED AT THE EARLY STAGES OF MYOGENESIS

SummaryMyogenic cell lines have been used extensively in the study of skeletal muscle development, regeneration, and homeostasis. To induce myogenic differentiation, culture media composed of a wide variety of growth factors and other additives have been used. Because the diversity in these components may modulate the differentiation process differentially, we describe a differentiation protocol that does not require the introduction of any factors to the differentiation media (DM) other than those present in the growth media. By culturing C2C12 skeletal myocytes on a coating of diluted Matrigel, a soluble basement membrane, consisting of collagen IV, laminin, heparan sulfate proteoglycans, and entactin, myogenic differentiation was accomplished by mere serum reduction. Assessment of myotube formation, creatine kinase activity, myosin heavy chain-fast, and myogenin demonstrated that the kinetics and extent of myogenic differentiation were superior using this protocol, compared with a commonly used differentiation protocol, in which an extracellular matrix is not provided and the DM contains horse serum. In addition, the elevated transactivation of a troponin-I promoter reporter construct suggested that myogenesis was enhanced at the transcriptional level. Finally, assessment of genomic deoxyribonucleic acid content revealed that the Matrigel differentiation protocol resulted in lowered proliferation. This protocol may aid studies aimed at elucidating mechanisms of myogenic differentiation, where a homogeneous population of myotubes is preferred.

Pre-cachexia in patients with stages I–III non-small cell lung cancer: Systemic inflammation and functional impairment without activation of skeletal muscle ubiquitin proteasome system

Cachexia is a prevalent phenomenon of non-small cell lung cancer (NSCLC) which is responsible for increased mortality and deterioration of physical performance. Preclinical research indicates that systemic inflammation induces cachexia-related muscle wasting through muscular Nuclear Factor-kappa B (NF-κB) signaling and subsequent ubiquitin proteasome system (UPS)-mediated proteolysis. As these pathways could be a target for early intervention strategies, it needs to be elucidated whether increased activation of these pathways is already present in early stage NSCLC cachexia. The aim of the present study was therefore to assess muscular NF-κB and UPS activation in patients with NSCLC pre-cachexia. Sixteen patients with newly diagnosed stages I-III NSCLC having <10% weight loss and ten healthy controls were studied. Body composition, systemic inflammation and exercise capacity were assessed in all subjects and NF-κB and UPS activity in vastus lateralis muscle biopsies in a subset. Patients showed increased plasma levels of C-reactive protein (CRP) (P<0.001), soluble Tumor Necrosis Factor receptor 1 (sTNF-R1) (P<0.05), fibrinogen (P<0.001) and decreased levels of albumin (P<0.001). No changes in fat free body mass or skeletal muscle NF-κB and UPS activity were observed, while peak oxygen consumption ( [Formula: see text] ) was significantly decreased in patients compared with healthy controls. In conclusion, this exploratory study demonstrates significantly reduced exercise capacity in NSCLC pre-cachexia despite maintenance of muscle mass and unaltered indices of UPS activation. The absence of muscular NF-κB-dependent inflammatory signaling supports the notion that transition of systemic to local inflammation is required to initiate UPS-dependent muscle wasting characteristic for (experimental) cachexia.

Glycogen synthase kinase-3β is required for the induction of skeletal muscle atrophy

Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3β (GSK-3β), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3β using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3β suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3β inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3β dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3β enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3β.

Segregation of myoblast fusion and muscle-specific gene expression by distinct ligand-dependent inactivation of GSK-3β

Myogenic differentiation involves myoblast fusion and induction of muscle-specific gene expression, which are both stimulated by pharmacological (LiCl), genetic, or IGF-I-mediated GSK-3β inactivation. To assess whether stimulation of myogenic differentiation is common to ligand-mediated GSK-3β inactivation, myoblast fusion and muscle-specific gene expression were investigated in response to Wnt-3a. Moreover, crosstalk between IGF-I/GSK-3β/NFATc3 and Wnt/GSK-3β/β-catenin signaling was assessed. While both Wnt-3a and LiCl promoted myoblast fusion, muscle-specific gene expression was increased by LiCl, but not by Wnt-3a or β-catenin over-expression. Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity. In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I. These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation.

Hypoxia differentially regulates muscle oxidative fiber type and metabolism in a HIF-1α-dependent manner

Loss of skeletal muscle oxidative fiber types and mitochondrial capacity is a hallmark of chronic obstructive pulmonary disease and chronic heart failure. Based on in vivo human and animal studies, tissue hypoxia has been hypothesized as determinant, but the direct effect of hypoxia on muscle oxidative phenotype remains to be established. Hence, we determined the effect of hypoxia on in vitro cultured muscle cells, including gene and protein expression levels of mitochondrial components, myosin isoforms (reflecting slow-oxidative versus fast-glycolytic fibers), and the involvement of the regulatory PPAR/PGC-1α pathway. We found that hypoxia inhibits the PPAR/PGC-1α pathway and the expression of mitochondrial components through HIF-1α. However, in contrast to our hypothesis, hypoxia stimulated the expression of slow-oxidative type I myosin via HIF-1α. Collectively, this study shows that hypoxia differentially regulates contractile and metabolic components of muscle oxidative phenotype in a HIF-1α-dependent manner.