ATP-Induced Increase in Intracellular Calcium Levels and Subsequent Activation of mTOR as Regulators of Skeletal Muscle Hypertrophy.

Intracellular signaling pathways, including the mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK) pathway, are activated by exercise, and promote skeletal muscle hypertrophy. However, the mechanisms by which these pathways are activated by physiological stimulation are not fully understood. Here we show that extracellular ATP activates these pathways by increasing intracellular Ca2+ levels ([Ca2+]i), and promotes muscle hypertrophy. [Ca2+]i in skeletal muscle was transiently increased after exercise. Treatment with ATP induced the increase in [Ca2+]i through the P2Y2 receptor/inositol 1,4,5-trisphosphate receptor pathway, and subsequent activation of mTOR in vitro. In addition, the ATP-induced increase in [Ca2+]i coordinately activated Erk1/2, p38 MAPK and mTOR that upregulated translation of JunB and interleukin-6. ATP also induced an increase in [Ca2+]i in isolated soleus muscle fibers, but not in extensor digitorum longus muscle fibers. Furthermore, administration of ATP led to muscle hypertrophy in an mTOR- and Ca2+-dependent manner in soleus, but not in plantaris muscle, suggesting that ATP specifically regulated [Ca2+]i in slow muscles. These findings suggest that ATP and [Ca2+]i are important mediators that convert mechanical stimulation into the activation of intracellular signaling pathways, and point to the P2Y receptor as a therapeutic target for treating muscle atrophy.

Truncated dystrophin ameliorates the dystrophic phenotype of mdx mice by reducing sarcolipin-mediated SERCA inhibition.

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45-55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45-55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.

Repurposing the Selective Oestrogen Receptor Modulator Tamoxifen for the Treatment of Duchenne Muscular Dystrophy.

Drug discovery is a long, expensive and risky process. Evaluating drugs that have already been proved safe for use in humans and testing them for a new indication greatly reduces the time and monetary costs involved in finding treatments for life-threatening conditions. Here tamoxifen, a drug that is used for the treatment of breast cancer, is investigated in a mouse model of Duchenne muscular dystrophy. Tamoxifen was efficacious in countering the symptoms of the disease without affecting the underlying genetic cause. Based on these results, tamoxifen has been tested in other forms of muscle disease with success. Drug repurposing may not only be a cost-effective manner for treating a variety of diseases, it may also help us uncover common mechanisms between conditions that were previously thought to be unrelated.

Low-Intensity Training and the C5a Complement Antagonist NOX-D21 Rescue the mdx Phenotype through Modulation of Inflammation.

Inflammatory events occurring in dystrophic muscles contribute to the progression of Duchenne muscular dystrophy (DMD). Low-intensity training (LIT) attenuates the phenotype of mdx mice, an animal model for DMD. Therefore, we postulated that LIT could have anti-inflammatory properties. We assessed levels of inflammatory cytokines and infiltrated immune cells in gastrocnemius muscle of mdx mice after LIT. We detected high levels of complement component C5a, chemokine ligand (CCL) 2, CD68+ monocytes/macrophages, and proinflammatory M1 macrophages in muscles of mdx mice. LIT decreased CCL2 levels, increased CD68+ cell numbers, and shifted the macrophage population to the regenerative M2 type. We investigated whether inhibition of C5a or CCL2 with L-aptamers could mimic the effects of LIT. Although no effect of CCL2 inhibition was detected, treatment with the C5a inhibitor, NOX-D21, rescued the phenotype of nonexercised mdx mice, but not of exercised ones. In both cases, the level of CD68+ cells increased and macrophage populations leaned toward the inflammatory M1 type. In muscles of nonexercised treated mice, the level of IL-1 receptor antagonist increased, damage decreased, and fibers were switched toward the glycolytic fast type; in muscles of exercised mice, fibers were switched to the oxidative slow type. These results reveal the effects of LIT on the inflammatory status of mdx mice and suggest that NOX-D21 could be an anti-inflammatory drug for DMD.

Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs (miR-1, miR-133a, and miR-206) in C2C12 Myocytes and mdx Mice.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs), is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion) into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration.

The Swiss Society of Experimental Pharmacology in Times of Change.

Experimental pharmacology is undergoing fundamental changes. This article describes the challenges and opportunities associated with these changes from the perspective of the Swiss Society of Pharmacology (SSEP), the society which aims to advance experimental pharmacology in Switzerland and abroad.

Caloric restriction induces energy-sparing alterations in skeletal muscle contraction, fiber composition and local thyroid hormone metabolism that persist during catch-up fat upon refeeding.

Weight regain after caloric restriction results in accelerated fat storage in adipose tissue. This catch-up fat phenomenon is postulated to result partly from suppressed skeletal muscle thermogenesis, but the underlying mechanisms are elusive. We investigated whether the reduced rate of skeletal muscle contraction-relaxation cycle that occurs after caloric restriction persists during weight recovery and could contribute to catch-up fat. Using a rat model of semistarvation-refeeding, in which fat recovery is driven by suppressed thermogenesis, we show that contraction and relaxation of leg muscles are slower after both semistarvation and refeeding. These effects are associated with (i) higher expression of muscle deiodinase type 3 (DIO3), which inactivates tri-iodothyronine (T3), and lower expression of T3-activating enzyme, deiodinase type 2 (DIO2), (ii) slower net formation of T3 from its T4 precursor in muscles, and (iii) accumulation of slow fibers at the expense of fast fibers. These semistarvation-induced changes persisted during recovery and correlated with impaired expression of transcription factors involved in slow-twitch muscle development. We conclude that diminished muscle thermogenesis following caloric restriction results from reduced muscle T3 levels, alteration in muscle-specific transcription factors, and fast-to-slow fiber shift causing slower contractility. These energy-sparing effects persist during weight recovery and contribute to catch-up fat.

Comparison of Experimental Protocols of Physical Exercise for mdx Mice and Duchenne Muscular Dystrophy Patients.

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene coding for dystrophin and leads to muscle degeneration, wheelchair dependence and death by cardiac or respiratory failure. Physical exercise has been proposed as a palliative therapy for DMD to maintain muscle strength and prevent contractures for as long as possible. However, its practice remains controversial because the benefits of training may be counteracted by muscle overuse and damage.The effects of physical exercise have been investigated in muscles of dystrophin-deficient mdx mice and in patients with DMD. However, a lack of uniformity among protocols limits comparability between studies and translatability of results from animals to humans. In the present review, we summarize and discuss published protocols used to investigate the effects of physical exercise on mdx mice and DMD patients, with the objectives of improving comparability between studies and identifying future research directions.

Diapocynin, a dimer of the NADPH oxidase inhibitor apocynin, reduces ROS production and prevents force loss in eccentrically contracting dystrophic muscle.

Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches.

Pharmacological prospects in the treatment of Duchenne muscular dystrophy.

PURPOSE OF REVIEW: The most encouraging recent advances regarding pharmacological agents for treating Duchenne muscular dystrophy (DMD) are summarized. Emphasis is given to compounds acting downstream of dystrophin, the protein lacking in DMD, on cellular pathways leading to pathological consequences. The author highlights the progress that may have the greatest potential for clinical use in DMD. RECENT FINDINGS: Modifying the transcripts of the mutated gene by exon skipping has led to expression of shortened dystrophins in DMD patients. Currently, the most promising potential drugs are the exon-skipping agents eteplirsen and drisapersen. Biglycan and SMTC1100 upregulate utrophin. The steroid receptor modulating compounds VBP15 and tamoxifen, and specific antioxidants appear promising agents for symptomatic therapy. SUMMARY: The past 18 months have seen a strong increase in the number of exciting reports on novel therapeutic agents for DMD. Exon-skipping agents have been fine-tuned to improve tissue delivery and stability. Impressive discoveries regarding pathogenic events in cellular signalling have revealed targets that were unknown in the context of DMD, thus enabling approaches that limit inflammation, fibrosis and necrosis. The targets are nuclear hormone receptors, NADPH-oxidases and Ca channels. Inhibition of NF-KB, transforming growth factor-alpha (TGF-α) and transforming growth factor-beta (TGF-ß)/myostatin production or action are also promising routes in counteracting the complex pathogenesis of DMD.

Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3ß. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

Epigallocatechin-3-gallate: a useful, effective and safe clinical approach for targeted prevention and individualised treatment of neurological diseases?

Neurodegenerative disorders show an increasing prevalence in a number of highly developed countries. Often, these diseases require life-long treatment mostly with drugs which are costly and mostly accompanied by more or less serious side-effects. Their heterogeneous manifestation, severity and outcome pose the need for individualised treatment options. There is an intensive search for new strategies not only for treating but also for preventing these diseases. Green tea and green tea extracts seem to be such a promising and safe alternative. However, data regarding the beneficial effects and possible underlying mechanism, specifically in clinical trials, are rare and rather controversial or non-conclusive. This review outlines the existing evidence from preclinical studies (cell and tissue cultures and animal models) and clinical trials regarding preventive and therapeutic effects of epigallcatechin-3-gallate in neurodegenerative diseases and considers antioxidative vs. pro-oxidative properties of the tea catechin important for dosage recommendations.

The anticancer drug tamoxifen counteracts the pathology in a mouse model of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe disorder characterized by progressive muscle wasting,respiratory and cardiac impairments, and premature death. No treatment exists so far, and the identification of active substances to fight DMD is urgently needed. We found that tamoxifen, a drug used to treat estrogen-dependent breast cancer, caused remarkable improvements of muscle force and of diaphragm and cardiac structure in the mdx(5Cv) mouse model of DMD. Oral tamoxifen treatment from 3 weeks of age for 15 months at a dose of 10 mg/kg/day stabilized myofiber membranes, normalized whole body force, and increased force production and resistance to repeated contractions of the triceps muscle above normal values. Tamoxifen improved the structure of leg muscles and diminished cardiac fibrosis by~ 50%. Tamoxifen also reduced fibrosis in the diaphragm, while increasing its thickness,myofiber count, and myofiber diameter, thereby augmenting by 72% the amount of contractile tissue available for respiratory function. Tamoxifen conferred a markedly slower phenotype to the muscles.Tamoxifen and its metabolites were present in nanomolar concentrations in plasma and muscles,suggesting signaling through high-affinity targets. Interestingly, the estrogen receptors ERa and ERb were several times more abundant in dystrophic than in normal muscles, and tamoxifen normalized the relative abundance of ERb isoforms. Our findings suggest that tamoxifen might be a useful therapy for DMD.

The effect of levofloxacin and moxifloxacin on cardiovascular functions of rats with streptozotocin-induced diabetes.

Fluoroquinolone antibiotics cause rare, but clinically important, adverse events including hyperglycaemia and hypoglycaemia. The present study focuses on the possible effect of levofloxacin and moxifloxacin on the cardiovascular functions of rats with type I diabetes. Both antibiotics caused bradycardia. Levofloxacin but not moxifloxacin caused hypoglycaemia in diabetic rats and an increase in amplitude of the ST segment revealed by electrocardiogram (ECG) analysis of isolated hearts. In pressurized mesenteric arteries, levofloxacin did not affect the endothelium-derived hyperpolarising factor (EDHF) pathway or its main components, the small-conductance Ca(2+) activated potassium (SK(Ca)) and intermediate-conductance Ca(2+) activated potassium (IK(Ca)) channels. In moxifloxacin-treated rats, an increase in the EDHF response was observed, which was largely attributed to SK(Ca)-activation. In conclusion, levofloxacin and moxifloxacin use appeared to vary but with no evidence of impairment of the cardiovascular function. However, it is still possible that these antibiotics may produce different effects if there are co-morbidities and therefore their use must be with care.

Activation of calcium signaling through Trpv1 by nNOS and peroxynitrite as a key trigger of skeletal muscle hypertrophy.

Skeletal muscle atrophy occurs in aging and pathological conditions, including cancer, diabetes and AIDS. Treatment of atrophy is based on either preventing protein-degradation pathways, which are activated during atrophy, or activating protein-synthesis pathways, which induce muscle hypertrophy. Here we show that neuronal nitric oxide synthase (nNOS) regulates load-induced hypertrophy by activating transient receptor potential cation channel, subfamily V, member 1 (TRPV1). The overload-induced hypertrophy was prevented in nNOS-null mice. nNOS was transiently activated within 3 min after overload. This activation promoted formation of peroxynitrite, a reaction product of nitric oxide with superoxide, which was derived from NADPH oxidase 4 (Nox4). Nitric oxide and peroxynitrite then activated Trpv1, resulting in an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) that subsequently triggered activation of mammalian target of rapamycin (mTOR). Notably, administration of the TRPV1 agonist capsaicin induced hypertrophy without overload and alleviated unloading- or denervation-induced atrophy. These findings identify nitric oxide, peroxynitrite and [Ca(2+)](i) as the crucial mediators that convert a mechanical load into an intracellular signaling pathway and lead us to suggest that TRPV1 could be a new therapeutic target for treating muscle atrophy.

Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration.

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.

Quantitative evaluation of the beneficial effects in the mdx mouse of epigallocatechin gallate, an antioxidant polyphenol from green tea.

In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (-)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients.