Altered skeletal muscle function and beneficial effects of exercise training in a rat model of induced pulmonary emphysema.

AIM: Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow obstruction and development of emphysema. Among the comorbidities associated with COPD, skeletal muscle dysfunction is known to affect exercise capacity and the survival rate of patients. Pulmonary rehabilitation (PR), via exercise training, is essential for COPD patients. However, the response to PR is most often moderate. An animal model that recapitulates critical features of chronic human disease and provides access to muscle function should therefore be useful to improve PR benefits. METHODS: We used a rat model of induced emphysema based on pulmonary instillations of elastase (ELA) and lipopolysaccharides (LPS). We assessed the long-term effects of ELA/LPS and the potential effectiveness of endurance training on the skeletal muscle function. In vivo strength of the animals, and ex vivo contractility, endurance, type 1 fiber proportion, fiber cross-sectional area, and capillarization of both soleus and extensor digitorum longus (EDL) were assessed. RESULTS: An impaired overall muscle strength with decreased force, reduced capillarization, and atrophy of type 1 fiber of EDL was observed in ELA/LPS rats. Soleus was not affected. Endurance training was able to reduce fatigability, and increase type 1 fiber proportion and capillarization of soleus, and improve force, endurance, and capillarization of EDL in control and ELA/LPS rats. CONCLUSION: Our rat model of induced emphysema, which shares some features with the phenotype present in patients with COPD, could represent a suitable model to study skeletal muscle dysfunction and the effects of exercise training on muscle function in patients.

Muscle strength, quantity and quality and muscle fat quantity and their association with oxidative stress in patients with facioscapulohumeral muscular dystrophy: Effect of antioxidant supplementation.

The purpose of this study was to identify causes of quadriceps muscle weakness in facioscapulohumeral muscular dystrophy (FSHD). To this aim, we evaluated quadriceps muscle and fat volumes by magnetic resonance imaging and their relationships with muscle strength and oxidative stress markers in adult patients with FSHD (n = 32) and healthy controls (n = 7), and the effect of antioxidant supplementation in 20 of the 32 patients with FSHD (n = 10 supplementation and n = 10 placebo) (NCT01596803). Compared with healthy controls, the dominant quadriceps strength and quality (muscle strength per unit of muscle volume) were decreased in patients with FSHD. In addition, fat volume was increased, without changes in total muscle volume. Moreover, in patients with FSHD, the lower strength of the non-dominant quadriceps was associated with lower muscle quality compared with the dominant muscle. Antioxidant supplementation significantly changed muscle and fat volumes in the non-dominant quadriceps, and muscle quality in the dominant quadriceps. This was associated with improved muscle strength (both quadriceps) and antioxidant response. These findings suggest that quadriceps muscle strength decline may not be simply explained by atrophy and may be influenced also by the muscle intrinsic characteristics. As FSHD is associated with increased oxidative stress, supplementation might reduce oxidative stress and increase antioxidant defenses, promoting changes in muscle function.

Response to Electrostimulation Is Impaired in Muscle Cells from Patients with Chronic Obstructive Pulmonary Disease.

Among the comorbidities associated with chronic obstructive pulmonary disease (COPD), skeletal muscle weakness and atrophy are known to affect patient survival rate. In addition to muscle deconditioning, various systemic and intrinsic factors have been implicated in COPD muscle dysfunction but an impaired COPD muscle adaptation to contraction has never been extensively studied. We submitted cultured myotubes from nine healthy subjects and nine patients with COPD to an endurance-type protocol of electrical pulse stimulation (EPS). EPS induced a decrease in the diameter, covered surface and expression of MHC1 in COPD myotubes. Although the expression of protein degradation markers was not affected, expression of the protein synthesis marker mTOR was not induced in COPD compared to healthy myotubes after EPS. The expression of the differentiation markers p16INK4a and p21 was impaired, while expression of Myf5 and MyoD tended to be affected in COPD muscle cells in response to EPS. The expression of mitochondrial biogenesis markers PGC1α and MFN2 was affected and expression of TFAM and COX1 tended to be reduced in COPD compared to healthy myotubes upon EPS. Lipid peroxidation was increased and the expression of the antioxidant enzymes SOD2 and GPx4 was affected in COPD compared to healthy myotubes in response to EPS. Thus, we provide evidence of an impaired response of COPD muscle cells to contraction, which might be involved in the muscle weakness observed in patients with COPD.

COPD is deleterious for pericytes: implications during training-induced angiogenesis in skeletal muscle.

Improvements in skeletal muscle endurance and oxygen uptake are blunted in patients with chronic obstructive pulmonary disease (COPD), possibly because of a limitation in the muscle capillary oxygen supply. Pericytes are critical for capillary blood flow adaptation during angiogenesis but may be impaired by COPD systemic effects, which are mediated by circulating factors. This study compared the pericyte coverage of muscle capillaries in response to 10 wk of exercise training in patients with COPD and sedentary healthy subjects (SHS). Fourteen patients with COPD were compared with seven matched SHS. SHS trained at moderate intensity corresponding to an individualized moderate-intensity patient with COPD trained at the same relative (%V̇o2: COPD-RI) or absolute (mL·min-1·kg-1: COPD-AI) intensity as SHS. Capillary-to-fiber ratio (C/F) and NG2+ pericyte coverage were assessed from vastus lateralis muscle biopsies, before and after 5 and 10 wk of training. We also tested in vitro the effect of COPD and SHS serum on pericyte morphology and mesenchymal stem cell (MSC) differentiation into pericytes. SHS showed greater improvement in aerobic capacity (V̇o2VT) than both patients with COPD-RI and patients with COPD-AI (Group × Time: P = 0.004). Despite a preserved increase in the C/F ratio, NG2+ pericyte coverage did not increase in patients with COPD in response to training, contrary to SHS (Group × Time: P = 0.011). Conversely to SHS serum, COPD serum altered pericyte morphology (P < 0.001) and drastically reduced MSC differentiation into pericytes (P < 0.001). Both functional capacities and pericyte coverage responses to exercise training are blunted in patients with COPD. We also provide direct evidence of the deleterious effect of COPD circulating factors on pericyte morphology and differentiation.NEW & NOTEWORTHY This work confirms the previously reported impairment in the functional response to exercise training of patients with COPD compared with SHS. Moreover, it shows for the first time that pericyte coverage of the skeletal capillaries is drastically reduced in patients with COPD compared with SHS during training-induced angiogenesis. Finally, it provides experimental evidence that circulating factors are involved in the impaired pericyte coverage of patients with COPD.

Effects of a human microenvironment on the differentiation of human myoblasts.

Myogenic differentiation mechanisms are generally assessed using a murine cell line placed in low concentrations of an animal-derived serum. To more closely approximate in vivo pathophysiological conditions, recent studies have combined the use of human muscle cells with human serum. Nevertheless, the in vitro studies of the effects of a human microenvironment on the differentiation process of human myoblasts require the identification of the culture conditions that would provide an optimal and reproducible differentiation process of human muscle cells. We assessed the differentiation variability resulting from the use of human myoblasts and serums from healthy subjects by measuring the myotube diameter, fusion index and surface covered by myotubes. We showed the preserved cell-dependent variability of the differentiation response of myoblasts cultured in human serums compared to FBS. We found that using a pool of serums reduced the serum-dependent variability of the myogenic response compared to individual serums. We validated our methodology by showing the atrophying effect of pooled serums from COPD patients on healthy human myotubes. By replacing animal-derived tissues with human myoblasts and serums, and by validating the sensitivity of cultured human muscle cells to a pathological microenvironment, this human cell culture model offers a valuable tool for studying the role of the microenvironment in chronic disease.

Impaired training-induced angiogenesis process with loss of pericyte-endothelium interactions is associated with an abnormal capillary remodelling in the skeletal muscle of COPD patients.

Chronic obstructive pulmonary disease (COPD) is associated with exercise intolerance and limits the functional gains in response to exercise training in patients compared to sedentary healthy subjects (SHS). The blunted skeletal muscle angiogenesis previously observed in COPD patients has been linked to these limited functional improvements, but its underlying mechanisms, as well as the potential role of oxidative stress, remain poorly understood. Therefore, we compared ultrastructural indexes of angiogenic process and capillary remodelling by transmission electron microscopy in 9 COPD patients and 7 SHS after 6 weeks of individualized moderate-intensity endurance training. We also assessed oxidative stress by plasma-free and esterified isoprostane (F2-IsoP) levels in both groups. We observed a capillary basement membrane thickening in COPD patients only (p = 0.008) and abnormal variations of endothelial nucleus density in response to exercise training in these patients when compared to SHS (p = 0.042). COPD patients had significantly fewer occurrences of pericyte/endothelium interdigitations, a morphologic marker of capillary maturation, than SHS (p = 0.014), and significantly higher levels of F2-IsoP (p = 0.048). Last, the changes in pericyte/endothelium interdigitations and F2-IsoP levels in response to exercise training were negatively correlated (r = - 0.62, p = 0.025). This study is the first to show abnormal capillary remodelling and to reveal impairments during the whole process of angiogenesis (capillary creation and maturation) in COPD patients. TRIAL REGISTRATION: NCT01183039 & NCT01183052, both registered 7 August 2010 (retrospectively registered).

Interactive whiteboard use in clinical reasoning sessions to teach diagnostic test ordering and interpretation to undergraduate medical students.

BACKGROUND: Over-testing of patients is a significant problem in clinical medicine that can be tackled by education. Clinical reasoning learning (CRL) is a potentially relevant method for teaching test ordering and interpretation. The feasibility might be improved by using an interactive whiteboard (IWB) during the CRL sessions to enhance student perceptions and behaviours around diagnostic tests. Overall, IWB/CRL could improve their skills. METHODS: Third-year undergraduate medical students enrolled in a vertically integrated curriculum were randomized into two groups before clinical placement in either a respiratory disease or respiratory physiology unit: IWB-based CRL plus clinical mentoring (IWB/CRL + CM: n = 40) or clinical mentoring only (CM-only: n = 40). Feasibility and learning outcomes were assessed. In addition, feedback via questionnaire of the IWB students and their classmates (n = 233) was compared. RESULTS: Analyses of the IWB/CRL sessions (n = 40, 27 paperboards) revealed that they met validated learning objectives. Students perceived IWB as useful and easy to use. After the IWB/CRL + CM sessions, students mentioned more hypothesis-based indications in a test ordering file (p <  0.001) and looked for more nonclinical signs directly on raw data tests (p <  0.01) compared with students in the CM-only group. Last, among students who attended pre- and post-assessments (n = 23), the number of diagnostic tests ordered did not change in the IWB/CRL + CM group (+ 7%; p = N.S), whereas it increased among CM-only students (+ 30%; p <  0.001). Test interpretability increased significantly in the IWB/CRL + CM group (from 4.7 to 37.2%; p <  0.01) but not significantly in the CM-only group (from 2.4 to 9.8%; p = 0.36). CONCLUSIONS: Integrating IWB into CRL sessions is feasible to teach test ordering and interpretation to undergraduate students. Moreover, student feedback and prospective assessment suggested a positive impact of IWB/CRL sessions on students' learning.

Oxidative stress regulates autophagy in cultured muscle cells of patients with chronic obstructive pulmonary disease.

The proteolytic autophagy pathway is enhanced in the lower limb muscles of patients with chronic obstructive pulmonary disease (COPD). Reactive oxygen species (ROS) have been shown to regulate autophagy in the skeletal muscles, but the role of oxidative stress in the muscle autophagy of patients with COPD is unknown. We used cultured myoblasts and myotubes from the quadriceps of eight healthy subjects and twelve patients with COPD (FEV1% predicted: 102.0% and 32.0%, respectively; p < 0.0001). We compared the autophagosome formation, the expression of autophagy markers, and the autophagic flux in healthy subjects and the patients with COPD, and we evaluated the effects of the 3-methyladenine (3-MA) autophagy inhibitor on the atrophy of COPD myotubes. Autophagy was also assessed in COPD myotubes treated with an antioxidant molecule, ascorbic acid. Autophagosome formation was increased in COPD myoblasts and myotubes (p = 0.011; p < 0.001), and the LC3 2/LC3 1 ratio (p = 0.002), SQSTM1 mRNA and protein expression (p = 0.023; p = 0.007), BNIP3 expression (p = 0.031), and autophagic flux (p = 0.002) were higher in COPD myoblasts. Inhibition of autophagy with 3-MA increased the COPD myotube diameter (p < 0.001) to a level similar to the diameter of healthy subject myotubes. Treatment of COPD myotubes with ascorbic acid decreased ROS concentration (p < 0.001), ROS-induced protein carbonylation (p = 0.019), the LC3 2/LC3 1 ratio (p = 0.037), the expression of SQSTM1 (p < 0.001) and BNIP3 (p < 0.001), and increased the COPD myotube diameter (p < 0.001). Thus, autophagy signaling is enhanced in cultured COPD muscle cells. Furthermore, the oxidative stress level contributes to the regulation of autophagy, which is involved in the atrophy of COPD myotubes in vitro.

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.

Erythropoietin protects against local anesthetic myotoxicity during continuous regional analgesia.

BACKGROUND: Local anesthetics offer the benefits of extended analgesia with greater patient satisfaction and faster rehabilitation compared with intravenous morphine. These benefits, however, can be offset by adverse iatrogenic muscle pain caused by bupivacaine. Here, the authors describe the mechanisms of local anesthetic-induced myotoxicity and a partial protective effect of recombinant human erythropoietin (rhEPO). METHODS: The authors developed a rat analgesia model with femoral nerve catheter and a cell culture model of human skeletal muscle myoblasts to study local anesthetic effects. Rats were randomly assigned to four different groups: daily intraperitoneal injection with 5,000 U/kg rhEPO or saline coupled to a perineural catheter injection with 1 ml/kg bupivacaine, 0.25%, or saline. In psoas rat muscle, oxygen consumption rates were measured using a Clark-type electrode in saponin-skinned fibers. Mitochondrial adenosine triphosphate synthesis rates were determined by bioluminescence. Enzymatic activity of mitochondrial respiratory chain complexes was measured on tissue homogenates using spectrophotometric procedures, and mitochondrial morphology was analyzed by transmission electron microscopy. In addition, the interaction between bupivacaine and rhEPO was investigated on human skeletal muscle myoblasts by fluorescence microscopy using mitotracker green and using the lipophilic cation JC-1. RESULTS: Bupivacaine caused impairment of mitochondrial structure and bioenergetics in rats. Human myoblasts treated with bupivacaine showed a dose-dependent decrease in mitochondrial membrane potential associated with unusual morphologies. Impairment of mitochondrial bioenergetics was prevented partially by the use of rhEPO coadministered with bupivacaine. CONCLUSIONS: The authors demonstrated a dose- and time-dependent protective effect of rhEPO against bupivacaine-induced myotoxicity in regional analgesia.