Guglielmo Duranti

Influence of the PDE5 inhibitor tadalafil on redox status and antioxidant defense system in C2C12 skeletal muscle cells

Phosphodiesterase type 5 inhibitors (PDE5Is), widely known for their beneficial effects onto male erectile dysfunction, seem to exert favorable effects onto metabolism as well. Tadalafil exposure increases oxidative metabolism of C2C12 skeletal muscle cells. A rise in fatty acid (FA) metabolism, requiring more oxygen, could induce a larger reactive oxygen species (ROS) release as a byproduct thus leading to a redox imbalance. The aim of this study was to determine how PDE5I tadalafil influences redox status in skeletal muscle cells to match the increasing oxidative metabolism. To this purpose, differentiated C2C12 skeletal muscle cells were treated with tadalafil and analyzed for total antioxidant capacity (TAC) and glutathione levels as marker of redox status; enzyme activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) engaged in antioxidant defense; and lipid peroxidation (TBARS) and protein carbonyls (PrCar) as markers of oxidative damage. Tadalafil increased total intracellular glutathione (tGSH), CAT, SOD, and GPx enzymatic activities while no changes were found in TAC. A perturbation of redox status, as showed by the decrease in the ratio between reduced/oxidized glutathione (GSH/GSSG), was observed. Nevertheless, it did not cause any change in TBARS and PrCar levels probably due to the enhancement in the antioxidant enzymatic network. Taken together, these data indicate that tadalafil, besides improving oxidative metabolism, may be beneficial to skeletal muscle cells by enhancing the enzymatic antioxidant system capacity.

Effects of Salmeterol on Skeletal Muscle Cells: Metabolic and Proapoptotic Features

PURPOSEnSalmeterol is a β2-adrenergic receptor agonist widely used for the treatment of asthma and chronic obstructive pulmonary disease. It has been shown that salmeterol is also used at supratherapeutic doses as performance-enhancing substance in sport practice. Although the abuse of β-agonists might determine some adverse effects, the molecular effects of salmeterol on skeletal muscle cells remain unclear.nnnMETHODSnWe evaluated the effects of salmeterol (0.1-10 μM) on both proliferative and differentiated rat L6C5 and mouse C2C12 skeletal muscle cell lines. The metabolic effects were evaluated by glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase, citrate synthase, 3-OH acyl-CoA dehydrogenase, and alanine transglutaminase activities. Cytotoxic and apoptotic effects were analyzed by 3-(4,5-dimethylthiazol-1)-5-(3-carboxymeth-oxyphenyl)-2H-tetrazolium, trypan blue exclusion assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Western blot analysis, and immunofluorescence staining.nnnRESULTSnWe showed that salmeterol reduced the growth rate of proliferating cells in a dose- and time-dependent manner (6-48 h). An increase in oxidative metabolism was found after 6 h in C2C12 and L6C5 myoblasts and in C2C12 myotubes with respect to control cells, while in L6C5 myotubes, anaerobic metabolism prevailed. Exposure of myoblasts and myotubes for 48 and 72 h at high salmeterol concentrations induced apoptosis by the activation of the intrinsic apoptotic pathway, as confirmed by the modulation of the apoptotic proteins Bcl-xL, caspase-9, and poly (ADP-ribose) polymerase and by the cytoplasmic release of Smac/DIABLO.nnnCONCLUSIONSnAltogether, our results demonstrate that short-term supratherapeutic salmeterol exposure increased oxidative metabolic pathways on skeletal muscle cells, whereas prolonged treatment inhibits cell growth and exerts either a cytostatic or a proapoptotic effect in a time- and dose-dependent way.

Chronic consumption of quercetin reduces erythrocytes oxidative damage: Evaluation at resting and after eccentric exercise in humans

The polyphenolic flavonoid quercetin has been shown to be a powerful antioxidant, in vitro and in murine models. However, its effect on redox status has been poorly examined in humans, particularly in combination with strenuous exercise. We hypothesized that quercetin supplementation would beneficially affect redox homeostasis in healthy individuals undergoing eccentric exercise. To test this hypothesis, the effects of chronic consumption of quercetin on glutathione system (reduced, oxidized, and reduced to oxidized glutathione ratio), oxidative damage [thiobarbituric acid reactive substances (TBARs)], antioxidant enzymatic network (catalase, glutathione peroxidase, superoxide dismutase) and resistance to lysis, were investigated in erythrocytes, a traditional model widely used to study the effects of oxidative stress as well as the protective effects of antioxidants. In a two weeks controlled, randomized, crossover, intervention trial, 14 individuals ingested 2 caps (1 g/d) of quercetin or placebo. Blood samples were collected before, after 2 weeks of supplementation and after a bout of eccentric exercise. Quercetin, reduced significantly erythrocytes lipid peroxidation levels and the susceptibility to hemolysis induced by the free radical generator AAPH, while no differences in antioxidant enzyme activities and glutathione homeostasis were found between the two groups. After a single bout of eccentric exercise, quercetin supplementation improved redox status as assessed by reduced/oxidized glutathione ratio analysis and reduced TBARs levels both in erythrocytes and plasma. In conclusion, our study provides evidences that chronic quercetin supplementation has antioxidant potential prior to and after a strenuous eccentric exercise thus making the erythrocytes capable to better cope with an oxidative insult.

The acute effect of Quercetin on muscle performance following a single resistance training session

PurposeTo examine the effect of acute quercetin (Q) ingestion on neuromuscular function, biomarkers of muscle damage, and rate of perceived exertion (RPE) in response to an acute bout of resistance training.Methods10 young men (22.1u2009±u20091.8 years, 24.1u2009±u20093.1 BMI) participated in a randomized, double-blind, crossover study. Subjects consumed Q (1xa0g/day) or placebo (PLA) 3xa0h prior to a resistance training session which consisted of 3 sets of 8 repetitions at 80% of the one repetition maximum (1RM) completed bilaterally for eight different resistance exercises. Electromyographic (EMG) signals were recorded from the knee extensor muscles during maximal isometric (MVIC) and isokinetic voluntary contractions, and during an isometric fatiguing test. Mechanical and EMG signals, biomarkers of cell damage, and RPE score were measured PRE, immediately POST, and 24xa0h (blood indices only) following the resistance exercise.ResultsAfter a single dose of Q, the torque–velocity curve of knee extensors was enhanced and after the resistance exercise, subjects showed a lower MVIC reduction (Q: 0.91u2009±u20096.10%, PLA: 8.66u2009±u20095.08%) with a greater rate of torque development (+u200910.6%, pu2009<u20090.005) and neuromuscular efficiency ratio (+u200928.2%, pu2009<u20090.005). Total volume of the resistance exercises was significantly greater in Q (1691.10u2009±u2009376.71 kgxa0rep) compared to PLA (1663.65u2009±u2009378.85 kgxa0rep) (pu2009<u20090.05) with a comparable RPE score. No significant differences were found in blood marker between treatments.ConclusionsThe acute ingestion of Q may enhance the neuromuscular performance during and after a resistance training session.

A multi-biomarker analysis of the antioxidant efficacy of Parkinson's disease therapy

Substantial evidences suggest that reactive oxygen species participate in the normal aging process and in cancer and neurodegenerative age-related diseases. Parkinsons disease (PD), one of the most common oxidative stress-associated pathology in aging people, is treated with a standard pharmacological protocol consisting in a combined therapy l-dopa plus an inhibitor of dopa-decarboxylase, such as carbidopa. The therapy is well validated for the ability to restoring dopaminergic neurotransmission in PD patients, while l-dopa and carbidopa ability in modulating oxidative stress is currently under discussion. Our aim was to evaluate the impact of l-dopa and carbidopa on several biomarkers of exogenously-induced oxidative stress to validate the overall antioxidant effectiveness of the therapy. For this purpose we used peripheral blood lymphocytes from healthy donors treated in vitro with l-dopa and carbidopa and then challenged by different concentrations of H2O2. Glutathione (GSH, GSSG, GSH/GSSG), malondialdehyde (TBARs), protein carbonyls as well as DNA damage (8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and micronuclei (MN)), modulation was evaluated. Our results show that l-dopa, but not carbidopa, decreases the markers of lipid and protein oxidation and increases the total content of glutathione. Both l-dopa and carbidopa (alone or in combination) are able to counteract the formation of 8-oxodG and to reduce H2O2-induced micronuclei.

Skeletal Muscle Pathophysiology: The Emerging Role of Spermine Oxidase and Spermidine

Skeletal muscle comprises approximately 40% of the total body mass. Preserving muscle health and function is essential for the entire body in order to counteract chronic diseases such as type II diabetes, cardiovascular diseases, and cancer. Prolonged physical inactivity, particularly among the elderly, causes muscle atrophy, a pathological state with adverse outcomes such as poor quality of life, physical disability, and high mortality. In murine skeletal muscle C2C12 cells, increased expression of the spermine oxidase (SMOX) enzyme has been found during cell differentiation. Notably, SMOX overexpression increases muscle fiber size, while SMOX reduction was enough to induce muscle atrophy in multiple murine models. Of note, the SMOX reaction product spermidine appears to be involved in skeletal muscle atrophy/hypertrophy. It is effective in reactivating autophagy, ameliorating the myopathic defects of collagen VI-null mice. Moreover, spermidine treatment, if combined with exercise, can affect D-gal-induced aging-related skeletal muscle atrophy. This review hypothesizes a role for SMOX during skeletal muscle differentiation and outlines its role and that of spermidine in muscle atrophy. The identification of new molecular pathways involved in the maintenance of skeletal muscle health could be beneficial in developing novel therapeutic lead compounds to treat muscle atrophy.