Syrah Grape Polyphenol Extracts Protect Human Skeletal Muscle Cells from Oxidative and Metabolic Stress Induced by Excess of Palmitic Acid: Effect of Skin/Seed Ripening Stage.

Metabolic skeletal muscle (SM) dysfunction, triggered by increased oxidative stress and mitochondrial impairment, is a pivotal contributor to obesity-associated insulin resistance (IR). Addressing obesity and SM IR demands substantial lifestyle changes including regular exercise and dietary adjustments that are difficult to follow over time. This prompted exploration of alternative approaches. Grape polyphenols (GPPs) have demonstrated a positive impact on metabolism, although few studies have focused on SM. Since grape polyphenolic content and composition depend on tissue and ripening, we explored the antioxidant potential of GPPs from skin (Sk) and seeds (Sd) extracted before veraison (Bv) and at mature (M) stages, on palmitate-induced IR in primary human SM cells. Despite their important difference in polyphenol (PP) content: Sd-BvPP > Sd-MPP/Sk-BvPP > Sk-MPP, all extracts reduced lipid peroxidation by 44-60%, up-regulated the heme-oxygenase 1 protein level by 75-132% and mitochondrial activity by 47-68%. Contrary to the other extracts, which improved insulin response by 50%, Sd-BvPP did not. Our findings suggest that compounds other than stilbenoids or anthocyanin-type molecules, present only in grape Sk, could play an active role in regulating SM oxidative and metabolic stress and insulin sensitivity, paving the way for further exploration of novel bioactive compounds.

The exon junction complex is required for DMD gene splicing fidelity and myogenic differentiation.

Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already spliced mRNAs. Here we investigate the importance of EJC for the correct splicing of the 2.2-megabase-long human DMD pre-mRNA, which encodes dystrophin, an essential protein involved in cytoskeletal organization and cell signaling. Using targeted RNA-seq, we show that knock-down of the eIF4A3 and Y14 core components of EJC in a human muscle cell line causes an accumulation of mis-splicing events clustered towards the 3' end of the DMD transcript (Dp427m). This deregulation is conserved in the short Dp71 isoform expressed ubiquitously except in adult skeletal muscle and is rescued with wild-type eIF4A3 and Y14 proteins but not with an EJC assembly-defective mutant eIF4A3. MLN51 protein and EJC-associated ASAP/PSAP complexes independently modulate the inclusion of the regulated exons 71 and 78. Our data confirm the protective role of EJC in maintaining splicing fidelity, which in the DMD gene is necessary to preserve the function of the critical C-terminal protein-protein interaction domain of dystrophin present in all tissue-specific isoforms. Given the role of the EJC in maintaining the integrity of dystrophin, we asked whether the EJC could also be involved in the regulation of a mechanism as complex as skeletal muscle differentiation. We found that eIF4A3 knockdown impairs myogenic differentiation by blocking myotube formation. Collectively, our data provide new insights into the functional roles of EJC in human skeletal muscle.

A Bioassay-Guided Fractionation of Rosemary Leaf Extract Identifies Carnosol as a Major Hypertrophy Inducer in Human Skeletal Muscle Cells.

A good quality of life requires maintaining adequate skeletal muscle mass and strength, but therapeutic agents are lacking for this. We developed a bioassay-guided fractionation approach to identify molecules with hypertrophy-promoting effect in human skeletal muscle cells. We found that extracts from rosemary leaves induce muscle cell hypertrophy. By bioassay-guided purification we identified the phenolic diterpene carnosol as the compound responsible for the hypertrophy-promoting activity of rosemary leaf extracts. We then evaluated the impact of carnosol on the different signaling pathways involved in the control of muscle cell size. We found that activation of the NRF2 signaling pathway by carnosol is not sufficient to mediate its hypertrophy-promoting effect. Moreover, carnosol inhibits the expression of the ubiquitin ligase E3 Muscle RING Finger protein-1 that plays an important role in muscle remodeling, but has no effect on the protein synthesis pathway controlled by the protein kinase B/mechanistic target of rapamycin pathway. By measuring the chymotrypsin-like activity of the proteasome, we found that proteasome activity was significantly decreased by carnosol and Muscle RING Finger 1 inactivation. These results strongly suggest that carnosol can induce skeletal muscle hypertrophy by repressing the ubiquitin-proteasome system-dependent protein degradation pathway through inhibition of the E3 ubiquitin ligase Muscle RING Finger protein-1.

Skeletal Ryanodine Receptors Are Involved in Impaired Myogenic Differentiation in Duchenne Muscular Dystrophy Patients.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting following repeated muscle damage and inadequate regeneration. Impaired myogenesis and differentiation play a major role in DMD as well as intracellular calcium (Ca2+) mishandling. Ca2+ release from the sarcoplasmic reticulum is mostly mediated by the type 1 ryanodine receptor (RYR1) that is required for skeletal muscle differentiation in animals. The study objective was to determine whether altered RYR1-mediated Ca2+ release contributes to myogenic differentiation impairment in DMD patients. The comparison of primary cultured myoblasts from six boys with DMD and five healthy controls highlighted delayed myoblast differentiation in DMD. Silencing RYR1 expression using specific si-RNA in a healthy control induced a similar delayed differentiation. In DMD myotubes, resting intracellular Ca2+ concentration was increased, but RYR1-mediated Ca2+ release was not changed compared with control myotubes. Incubation with the RYR-calstabin interaction stabilizer S107 decreased resting Ca2+ concentration in DMD myotubes to control values and improved calstabin1 binding to the RYR1 complex. S107 also improved myogenic differentiation in DMD. Furthermore, intracellular Ca2+ concentration was correlated with endomysial fibrosis, which is the only myopathologic parameter associated with poor motor outcome in patients with DMD. This suggested a potential relationship between RYR1 dysfunction and motor impairment. Our study highlights RYR1-mediated Ca2+ leakage in human DMD myotubes and its key role in myogenic differentiation impairment. RYR1 stabilization may be an interesting adjunctive therapeutic strategy in DMD.

Human neuromuscular junction on micro-structured microfluidic devices implemented with a custom micro electrode array (MEA).

In the neuromuscular system, signal transmission from motor neurons (MNs) to innervated muscle fibers is crucial for their synaptic function, viability, and maintenance. In order to better understand human neuromuscular junction (hNMJ) functionality, it is important to develop on-a-chip devices with human cells. To investigate this cell network, microfluidic platforms are useful to grow different cell types in isolated compartments. Such devices have already been developed to study in vitro neuronal circuitry. Here, we combined microfluidics with two techniques: soft lithography and custom microelectrodes array (MEA). Our goal was to create hNMJs on a specific pattern of electrodes to stimulate pre-synaptic axons and record post-synaptic muscle activity. Micromachining was used to create structurations to guide muscle growth above electrodes, without impairing axon propagation, therefore optimizing the effectiveness of activity recording. Electrodes were also arranged to be aligned with the microfluidic chambers in order to specifically stimulate axons that were growing between the two compartments. Isolation of the two cell types allows for the selective treatment of neurons or muscle fibers to assess NMJ functionality hallmarks. Altogether, this microfluidic/microstructured/MEA platform allowed mature and functional in vitro hNMJ modelling. We demonstrate that electrical activation of MNs can trigger recordable extracellular muscle action potentials. This study provides evidence for a physiologically relevant model to mimic a hNMJ that will in the future be a powerful tool, more sensitive than calcium imaging, to better understand and characterize NMJs and their disruption in neurodegenerative diseases.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Identification of a Sesquiterpene Lactone from Arctium lappa Leaves with Antioxidant Activity in Primary Human Muscle Cells.

Many pathologies affecting muscles (muscular dystrophies, sarcopenia, cachexia, renal insufficiency, obesity, diabetes type 2, etc.) are now clearly linked to mechanisms involving oxidative stress. In this context, there is a growing interest in exploring plants to find new natural antioxidants to prevent the appearance and the development of these muscle disorders. In this study, we investigated the antioxidant properties of Arctium lappa leaves in a model of primary human muscle cells exposed to H2O2 oxidative stress. We identified using bioassay-guided purification, onopordopicrin, a sesquiterpene lactone as the main molecule responsible for the antioxidant activity of A. lappa leaf extract. According to our findings, onopordopicrin inhibited the H2O2-mediated loss of muscle cell viability, by limiting the production of free radicals and abolishing DNA cellular damages. Moreover, we showed that onopordopicrin promoted the expression of the nuclear factor-erythroid-2-related factor 2 (Nrf2) downstream target protein heme oxygenase-1 (HO-1) in muscle cells. By using siRNA, we demonstrated that the inhibition of the expression of Nrf2 reduced the protective effect of onopordopicrin, indicating that the activation of the Nrf2/HO-1 signaling pathway mediates the antioxidant effect of onopordopicrin in primary human muscle cells. Therefore, our results suggest that onopordopicrin may be a potential therapeutic molecule to fight against oxidative stress in pathological specific muscle disorders.

No Additive Effects of Polyphenol Supplementation and Exercise Training on White Adiposity Determinants of High-Fat Diet-Induced Obese Insulin-Resistant Rats.

One of the major insulin resistance instigators is excessive adiposity and visceral fat depots. Individually, exercise training and polyphenol intake are known to exert health benefits as improving insulin sensitivity. However, their combined curative effects on established obesity and insulin resistance need further investigation particularly on white adipose tissue alterations. Therefore, we compared the effects on different white adipose tissue depot alterations of a combination of exercise and grape polyphenol supplementation in obese insulin-resistant rats fed a high-fat diet to the effects of a high-fat diet alone or a nutritional supplementation of grape polyphenols (50 mg/kg/day) or exercise training (1 hr/day to 5 days/wk consisting of treadmill running at 32 m/min for a 10% slope), for a total duration of 8 weeks. Separately, polyphenol supplementation and exercise decreased the quantity of all adipose tissue depots and mesenteric inflammation. Exercise reduced adipocytes' size in all fat stores. Interestingly, combining exercise to polyphenol intake presents no more cumulative benefit on adipose tissue alterations than exercise alone. Insulin sensitivity was improved at systemic, epididymal, and inguinal adipose tissues levels in trained rats thus indicating that despite their effects on adipocyte morphological/metabolic changes, polyphenols at nutritional doses remain less effective than exercise in fighting insulin resistance.

Interest of colchicine in the treatment of acute myocardial infarct responsible for heart failure in a mouse model.

BACKGROUND: Inflammation is deeply involved in the pathophysiology of ischemia-reperfusion (I/R) lesions and ventricular remodeling due to an acute myocardial infarction (AMI). Colchicine as a pleiotropic anti-inflammatory molecule may exert cardioprotective effects under acute ischemia. Here, we aimed to evaluate the impact of colchicine on reperfusion injury in a mouse model. METHOD: Myocardial ischemia/reperfusion (I/R) injury was induced in C57BL/6 male mice, after 45min ligation of the left coronary artery followed by reperfusion. 400µg/kg of colchicine or the vehicle was administrated intraperitoneally (i.p.) 25min before the reperfusion (blinded administration). Mice were sacrificed at 24h after the acute myocardial ischemia (AMI) and the infarct size was determined. Circulating level of troponin and cytokines profile were assessed 4h after the AMI. An echocardiography was performed in a follow-up group mice, 48h and 8weeks after the AMI. RESULTS: The infarct size was reduced in colchicine treated mice (39.8±3.5% versus 52.9±3.2%, p<0.05). Troponin was significantly lower in the colchicine treated mice (7015.7±1423.7pg/mL, n=5 vs 30,723.7±7959.9pg/mL in the placebo group, n=6; p<0.0001). Fibrosis was decreased in the Colchicine group (24.51±3.13% vs 11.38±2.46%, p=0.03). In the follow-up group mice (n=8), there were no differences between mice treated with placebo (n=9) and mice treated with colchicine (n=9) regarding to cardiac remodeling parameters but outflow approximated by the ITV was higher in the colchicine group. CONCLUSION: In conclusion, colchicine allowed a significant reduction of infarct size in mice, improves hemodynamic parameters and decrease cardiac fibrosis.

Retinoic acid maintains human skeletal muscle progenitor cells in an immature state.

Muscle satellite cells are resistant to cytotoxic agents, and they express several genes that confer resistance to stress, thus allowing efficient dystrophic muscle regeneration after transplantation. However, once they are activated, this capacity to resist to aggressive agents is diminished resulting in massive death of transplanted cells. Although cell immaturity represents a survival advantage, the signalling pathways involved in the control of the immature state remain to be explored. Here, we show that incubation of human myoblasts with retinoic acid impairs skeletal muscle differentiation through activation of the retinoic-acid receptor family of nuclear receptor. Conversely, pharmacologic or genetic inactivation of endogenous retinoic-acid receptors improved myoblast differentiation. Retinoic acid inhibits the expression of early and late muscle differentiation markers and enhances the expression of myogenic specification genes, such as PAX7 and PAX3. These results suggest that the retinoic-acid-signalling pathway might maintain myoblasts in an undifferentiated/immature stage. To determine the relevance of these observations, we characterised the retinoic-acid-signalling pathways in freshly isolated satellite cells in mice and in siMYOD immature human myoblasts. Our analysis reveals that the immature state of muscle progenitors is correlated with high expression of several genes of the retinoic-acid-signalling pathway both in mice and in human. Taken together, our data provide evidences for an important role of the retinoic-acid-signalling pathway in the regulation of the immature state of muscle progenitors.

Grape polyphenols supplementation reduces muscle atrophy in a mouse model of chronic inflammation.

OBJECTIVES: Polyphenols (PP) have demonstrated beneficial effects on low-grade inflammation and oxidative stress; however, little is known about their effect on highly inflamed muscle. The purposes of this study were (i) to evaluate muscle alteration induced by high-grade inflammation, and (ii) to test the effects of red grape PP supplementation on these alterations. METHODS: We used a transgenic mice model (transforming growth factor [TGF] mice) to develop a high T cell-dependent inflammation and C57 BL/6 control (CTL) mice model. Skeletal muscles of TGF and CTL mice were investigated for inflammation, atrophy and oxidative stress markers. Isolated mitochondria from hindlimb muscles were used for respiration with pyruvate as substrate and oxidative damages were measured by Western blot. TGF mice were supplemented with a mixture of red grape polyphenols (50 mg/kg/d) for 4 wk. Data were analyzed by one-way analysis of variance (ANOVA) and post hoc Bonferroni's multiple comparison tests. RESULTS: TGF mice presented skeletal muscle inflammation, oxidative stress, mitochondrial alteration and muscle atrophy. Atrophy was associated with two distinct pathways: (i) one linked to inflammation, NF-κB activation and increased ubiquitin ligase expression, and (ii) one dependent on reactive oxygen species (ROS) production leading to damaged mitochondria accumulation and activation of caspase-9 and 3. Supplementation of TGF mice with a mixture of red grape polyphenols (50 mg/kg/d) for 4 wk improved mitochondrial function and highly decreased caspases activation, which allowed muscle atrophy mitigation. CONCLUSIONS: These observations suggest that nutritional dosages of red grape polyphenols might be beneficial for reducing skeletal muscle atrophy, even in a high-grade inflammation environment.

Effects of vitamin C, vitamin E, zinc gluconate, and selenomethionine supplementation on muscle function and oxidative stress biomarkers in patients with facioscapulohumeral dystrophy: a double-blind randomized controlled clinical trial.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease characterized by progressive weakness and atrophy of specific skeletal muscles. As growing evidence suggests that oxidative stress may contribute to FSHD pathology, antioxidants that might modulate or delay oxidative insults could help in maintaining FSHD muscle function. Our primary objective was to test whether oral administration of vitamin C, vitamin E, zinc gluconate, and selenomethionine could improve the physical performance of patients with FSHD. Adult patients with FSHD (n=53) were enrolled at Montpellier University Hospital (France) in a randomized, double-blind, placebo-controlled pilot clinical trial. Patients were randomly assigned to receive 500 mg vitamin C, 400mg vitamin E, 25mg zinc gluconate and 200 µg selenomethionine (n=26), or matching placebo (n=27) once a day for 17 weeks. Primary outcomes were changes in the two-minute walking test (2-MWT), maximal voluntary contraction, and endurance limit time of the dominant and nondominant quadriceps (MVCQD, MVCQND, TlimQD, and TlimQND, respectively) after 17 weeks of treatment. Secondary outcomes were changes in the antioxidant status and oxidative stress markers. Although 2-MWT, MVCQ, and TlimQ were all significantly improved in the supplemented group at the end of the treatment compared to baseline, only MVCQ and TlimQ variations were significantly different between groups (MVCQD: P=0.011; MVCQND: P=0.004; TlimQD: P=0.028; TlimQND: P=0.011). Similarly, the vitamin C (P<0.001), vitamin E as α-tocopherol (P<0.001), vitamin C/vitamin E ratio (P=0.017), vitamin E γ/α ratio (P=0.022) and lipid peroxides (P<0.001) variations were significantly different between groups. In conclusion, vitamin E, vitamin C, zinc, and selenium supplementation has no significant effect on the 2-MWT, but improves MVCQ and TlimQ of both quadriceps by enhancing the antioxidant defenses and reducing oxidative stress. This trial was registered at clinicaltrials.gov (number: NCT01596803).

Glutathione peroxidase 3, a new retinoid target gene, is crucial for human skeletal muscle precursor cell survival.

Protection of satellite cells from cytotoxic damages is crucial to ensure efficient adult skeletal muscle regeneration and to improve therapeutic efficacy of cell transplantation in degenerative skeletal muscle diseases. It is therefore important to identify and characterize molecules and their target genes that control the viability of muscle stem cells. Recently, we demonstrated that high aldehyde dehydrogenase activity is associated with increased viability of human myoblasts. In addition to its detoxifying activity, aldehyde dehydrogenase can also catalyze the irreversible oxidation of vitamin A to retinoic acid; therefore, we examined whether retinoic acid is important for myoblast viability. We showed that when exposed to oxidative stress induced by hydrogen peroxide, adherent human myoblasts entered apoptosis and lost their capacity for adhesion. Pre-treatment with retinoic acid reduced the cytotoxic damage ex vivo and enhanced myoblast survival in transplantation assays. The effects of retinoic acid were maintained in dystrophic myoblasts derived from facioscapulohumeral patients. RT-qPCR analysis of antioxidant gene expression revealed glutathione peroxidase 3 (Gpx3), a gene encoding an antioxidant enzyme, as a potential retinoic acid target gene in human myoblasts. Knockdown of Gpx3 using short interfering RNA induced elevation in reactive oxygen species and cell death. The anti-cytotoxic effects of retinoic acid were impaired in GPx3-inactivated myoblasts, which indicates that GPx3 regulates the antioxidative effects of retinoic acid. Therefore, retinoid status and GPx3 levels may have important implications for the viability of human muscle stem cells.

Functional muscle impairment in facioscapulohumeral muscular dystrophy is correlated with oxidative stress and mitochondrial dysfunction.

Facioscapulohumeral muscular dystrophy (FSHD), the most frequent muscular dystrophy, is an autosomal dominant disease. In most individuals with FSHD, symptoms are restricted to muscles of the face, arms, legs, and trunk. FSHD is genetically linked to contractions of the D4Z4 repeat array causing activation of several genes. One of these maps in the repeat itself and expresses the DUX4 (the double homeobox 4) transcription factor causing a gene deregulation cascade. In addition, analyses of the RNA or protein expression profiles in muscle have indicated deregulations in the oxidative stress response. Since oxidative stress affects peripheral muscle function, we investigated mitochondrial function and oxidative stress in skeletal muscle biopsies and blood samples from patients with FSHD and age-matched healthy controls, and evaluated their association with physical performances. We show that specifically, oxidative stress (lipid peroxidation and protein carbonylation), oxidative damage (lipofuscin accumulation), and antioxidant enzymes (catalase, copper-zinc-dependent superoxide dismutase, and glutathione reductase) were higher in FSHD than in control muscles. FSHD muscles also presented abnormal mitochondrial function (decreased cytochrome c oxidase activity and reduced ATP synthesis). In addition, the ratio between reduced (GSH) and oxidized glutathione (GSSG) was strongly decreased in all FSHD blood samples as a consequence of GSSG accumulation. Patients with FSHD also had reduced systemic antioxidative response molecules, such as low levels of zinc (a SOD cofactor), selenium (a GPx cofactor involved in the elimination of lipid peroxides), and vitamin C. Half of them had a low ratio of gamma/alpha tocopherol and higher ferritin concentrations. Both systemic oxidative stress and mitochondrial dysfunction were correlated with functional muscle impairment. Mitochondrial ATP production was significantly correlated with both quadriceps endurance (T(LimQ)) and maximal voluntary contraction (MVC(Q)) values (rho=0.79, P=0.003; rho=0.62, P=0.05, respectively). The plasma concentration of oxidized glutathione was negatively correlated with the T(LimQ), MVC(Q) values, and the 2-min walk distance (MWT) values (rho=-0.60, P=0.03; rho=-0.56, P=0.04; rho=-0.93, P<0.0001, respectively). Our data characterized oxidative stress in patients with FSHD and demonstrated a correlation with their peripheral skeletal muscle dysfunction. They suggest that antioxidants that might modulate or delay oxidative insult may be useful in maintaining FSHD muscle functions.

E2F transcription factor-1 deficiency reduces pathophysiology in the mouse model of Duchenne muscular dystrophy through increased muscle oxidative metabolism.

E2F1 deletion leads to increased mitochondrial number and function, increased body temperature in response to cold and increased resistance to fatigue with exercise. Since E2f1-/- mice show increased muscle performance, we examined the effect of E2f1 genetic inactivation in the mdx background, a mouse model of Duchenne muscular dystrophy (DMD). E2f1-/-;mdx mice demonstrated a strong reduction of physiopathological signs of DMD, including preservation of muscle structure, decreased inflammatory profile, increased utrophin expression, resulting in better endurance and muscle contractile parameters, comparable to normal mdx mice. E2f1 deficiency in the mdx genetic background increased the oxidative metabolic gene program, mitochondrial activity and improved muscle functions. Interestingly, we observed increased E2F1 protein levels in DMD patients, suggesting that E2F1 might represent a promising target for the treatment of DMD.

AMPK activation stimulates autophagy and ameliorates muscular dystrophy in the mdx mouse diaphragm.

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Adaptive support ventilation prevents ventilator-induced diaphragmatic dysfunction in piglet: an in vivo and in vitro study.

BACKGROUND: Contrary to adaptive support ventilation (ASV), prolonged totally controlled mechanical ventilation (CMV) results in the absence of diaphragm activity and causes ventilator-induced diaphragmatic dysfunction. Because maintaining respiratory muscles at rest is likely a major cause of ventilator-induced diaphragmatic dysfunction, ASV may prevent its occurrence in comparison with CMV. The aim of our study was to compare the effects of ASV with those of CMV on both in vivo and in vitro diaphragmatic properties. METHODS: Two groups of six anesthetized piglets were ventilated during a 72-h period. Piglets in the CMV group (n = 6) were ventilated without spontaneous ventilation, and piglets in the ASV group (n = 6) were ventilated with spontaneous breaths. Transdiaphragmatic pressure was measured after bilateral, supramaximal transjugular stimulation of the two phrenic nerves. A pressure-frequency curve was drawn after stimulation from 20 to 120 Hz of the phrenic nerves. Diaphragm fiber proportions and mean sectional area were evaluated. RESULTS: After 72 h of ventilation, transdiaphragmatic pressure decreased by 30% of its baseline value in the CMV group, whereas it did not decrease in the ASV group. Although CMV was associated with an atrophy of the diaphragm (evaluated by mean cross-sectional area of both the slow and fast myosin chains), atrophy was not detected in the ASV group. CONCLUSION: Maintaining diaphragmatic contractile activity by using the ASV mode may protect the diaphragm against the deleterious effect of prolonged CMV, as demonstrated both in vitro and in vivo, in healthy piglets.

Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle.

UNLABELLED: The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties. BACKGROUND: GNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods. CONCLUSION/SIGNIFICANCE: Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

L-arginine decreases inflammation and modulates the nuclear factor-kappaB/matrix metalloproteinase cascade in mdx muscle fibers.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disorder associated with dystrophin deficiency that results in chronic inflammation, sarcolemma damage, and severe skeletal muscle degeneration. Recently, the use of L-arginine, the substrate of nitric oxide synthase (nNOS), has been proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD. However, little is known about signaling events that occur in dystrophic muscle with l-arginine treatment. Considering the implication of inflammation in dystrophic processes, we asked whether L-arginine inhibits inflammatory signaling cascades. We demonstrate that L-arginine decreases inflammation and enhances muscle regeneration in the mdx mouse model. Classic stimulatory signals, such as proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, are significantly decreased in mdx mouse muscle, resulting in lower nuclear factor (NF)-kappaB levels and activity. NF-kappaB serves as a pivotal transcription factor with multiple levels of regulation; previous studies have shown perturbation of NF-kappaB signaling in both mdx and DMD muscle. Moreover, L-arginine decreases the activity of metalloproteinase (MMP)-2 and MMP-9, which are transcriptionally activated by NF-kappaB. We show that the inhibitory effect of L-arginine on the NF-kappaB/MMP cascade reduces beta-dystroglycan cleavage and translocates utrophin and nNOS throughout the sarcolemma. Collectively, our results clarify the molecular events by which L-arginine promotes muscle membrane integrity in dystrophic muscle and suggest that NF-kappaB-related signaling cascades could be potential therapeutic targets for DMD management.

Modulation of p38 mitogen-activated protein kinase cascade and metalloproteinase activity in diaphragm muscle in response to free radical scavenger administration in dystrophin-deficient Mdx mice.

Duchenne muscular dystrophy muscles undergo increased oxidative stress and altered calcium homeostasis, which contribute to myofiber loss by trigging both necrosis and apoptosis. Here, we asked whether treatment with free radical scavengers could improve the dystrophic pattern of mdx muscles. Five-week-old mdx mice were treated for 2 weeks with alpha-lipoic acid/l-carnitine. This treatment decreased the plasmatic creatine kinase level, the antioxidant enzyme activity, and lipid peroxidation products in mdx diaphragm. Free radical scavengers also modulated the phosphorylation/activity of some component of the mitogen-activated protein kinase (MAPK) cascades: p38 MAPK, the extracellular signal-related kinase, and the Jun kinase. beta-Dystroglycan (beta-DG), a multifunctional adaptor or scaffold capable of interacting with components of the extracellular signal-related kinase-MAP kinase cascade, was also affected after treatment. In the mdx muscles, beta-DG (43 kd) was cleaved by matrix metalloproteinases into a 30-kd form (beta-DG30). We show that the proinflammatory protein nuclear factor-kappaB activator decreased after the treatment, leading to a significant reduction of matrix metalloproteinase activity in the mdx diaphragm. Our data highlight the implication of oxidative stress and cell signaling defects in dystrophin-deficient muscle via the MAP kinase cascade-beta-DG interaction and nuclear factor-kappaB-mediated inflammation process.