Search

The most common diseases are of polygenic origin. Since early last century, these are being treated with drugs that act downstream of the genetic hurdle. This strategy has saved millions of lives. The advent of genetics revealed the molecular basis of diseases of monogenic origin, including the neuromuscular ones. It was tempting to believe that the defective genes can be repaired with novel chemicals, with a cure being “just down the road”.

In 2003, nuclear power received renewed interest as a perceived climate-neutral way to meet high energy demands of large industrialized countries, such as China, India, Russia and the USA. It triggered a growing demand for uranium (U) as nuclear fuel. Dubbed the 'nuclear renaissance', the U-price rose over tenfold before the global credit crisis dampend the rush. Many efforts to capitalise on the renewed demand focused on Africa. This paper provides an overview on the type and extent of uranium mining, production and exploration on the African continent and discusses the economic benefits as well as the potential environmental and health risks and the long-term needs for remediation of legacy sites. The actual historical results of uranium mining activities in more than thirty African countries provide data against which to assess the existing risks of uranium development. The already existing uraniferous waste in several African countries threatens scarce water resources and the health of adjacent residents. Responsibility should rest with the governments and the companies to ensure that these threats are not realized.

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.

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.

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.

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.

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.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.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-β)/myostatin production or action are also promising routes in counteracting the complex pathogenesis of DMD.

Background and Purpose Chronic elevation in intracellular Ca2+ concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca2+ overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca2+-independent form of PL A2 (iPLA2). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca2+ handling and muscle function in mdx mice. Experimental Approach Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca2+ influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca2+ concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. Key Results Dox at 1–30 μM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca2+ influx in myotubes. Stimulated elevations of Ca2+ concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. Conclusions and Implications Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.

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 Ca2+ activated potassium (SKCa) and intermediate-conductance Ca2+ activated potassium (IKCa) channels. In moxifloxacin-treated rats, an increase in the EDHF response was observed, which was largely attributed to SKCa-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.

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.

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.

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.

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx(5Cv) mouse, an animal model for DMD. Male mdx(5Cv) mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine(®) implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx(5Cv) mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force-frequency relationship of melatonin-treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD.

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.

Skeletal muscle contraction is reputed not to depend on extracellular Ca2+. Indeed, stricto sensu , excitation-contraction coupling does not necessitate entry of Ca2+. However, we previously observed that, during sustained activity (repeated contractions), entry of Ca2+is needed to maintain force production. In the present study, we evaluated the possible involvement of the canonical transient receptor potential (TRPC)1 ion channel in this entry of Ca2+and investigated its possible role in muscle function. Patch-clamp experiments reveal the presence of a small-conductance channel (13 pS) that is completely lost in adult fibers from TRPC1−/−mice. The influx of Ca2+through TRPC1 channels represents a minor part of the entry of Ca2+into muscle fibers at rest, and the activity of the channel is not store dependent. The lack of TRPC1 does not affect intracellular Ca2+concentration ([Ca2+]i) transients reached during a single isometric contraction. However, the involvement of TRPC1-related Ca2+entry is clearly emphasized in muscle fatigue. Indeed, muscles from TRPC1−/−mice stimulated repeatedly progressively display lower [Ca2+]itransients than those observed in TRPC1+/+fibers, and they also present an accentuated progressive loss of force. Interestingly, muscles from TRPC1−/−mice display a smaller fiber cross-sectional area, generate less force per cross-sectional area, and contain less myofibrillar proteins than their controls. They do not present other signs of myopathy. In agreement with in vitro experiments, TRPC1−/−mice present an important decrease of endurance of physical activity. We conclude that TRPC1 ion channels modulate the entry of Ca2+during repeated contractions and help muscles to maintain their force during sustained repeated contractions.

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca(2+) entry through sarcolemmal cationic channels. Using the Mn(2+) quench method, we show here that Mn(2+) entry triggered by Ca(2+) store depletion but not basal Mn(2+) entry relies on Ca(2+)-independent PLA(2) (iPLA(2)) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx(5cv) mouse. iPLA(2) was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA(2) lipid product lysophosphatidylcholine was found to trigger Ca(2+) entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA(2) and lysophospholipid production may be of interest to reduce Ca(2+) entry and subsequent degeneration of dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by the absence of the protein dystrophin. Because oxidative stress contributes to the pathogenesis of DMD, we investigated if a green tea polyphenol blend (GTP) and its major polyphenol (-)-epigallocatechin gallate (EGCg), could protect muscle cell primary cultures from oxidative damage induced by hydrogen peroxide (H(2)O(2)) in the widely used mdx mouse model. On-line fluorimetric measurements using an H(2)O(2)-sensitive probe indicated that GTP and EGCg scavenged peroxide in a concentration-dependent manner. A 48 h exposure to EGCg increased glutathione content but did not alter the expression of proteins involved in membrane stabilization and repair. Pretreatment of dystrophic cultures with GTP or EGCg 48 h before exposure to H(2)O(2) improved cell survival. Normal cultures were protected by GTP but not by EGCg. 67LR, a receptor for EGCg, was seven times more abundant in dystrophic compared with normal cultures. Altogether our results demonstrate that GTP and EGCg protect muscle cells by scavenging H(2)O(2) and by improving the glutathione balance. In addition, the higher levels of 67LR in dystrophic muscle cells compared with normal ones likely contribute to EGCg-mediated survival.

Background and purpose: Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by the absence of the cytoskeletal protein dystrophin. This leads to muscle cell death accompanied by chronic inflammation. Cyclosporin A (CsA) is a powerful immunosuppressive drug, which has been proposed for DMD treatment. CsA also directly regulates the mitochondrial permeability transition pore (mPTP), which participates in cell death pathways through the inhibition of cyclophilin D. Here, we evaluated whether Debio 025, a cyclophilin inhibitor with no immunosuppressive activity, improves the dystrophic condition in a mouse model of DMD, through regulation of mPTP. Experimental approach: The potency of Debio 025 to protect mouse dystrophic cells against mitochondria-mediated death was assessed by caspase-3 activity and calcium retention capacity assays. Mdx5Cv mice (3-week-old) were treated daily by gavage for 2 weeks with Debio 025 (10, 30 or 100 mg kg−1), CsA (10 mg kg−1) or placebo. The effects on muscle necrosis and function were measured. Key results: In vitro investigations showed protective effect of low concentrations of Debio 025 against cell death. Histology demonstrated that Debio 025 partially protected the diaphragm and soleus muscles against necrosis (10 and 100 mg kg−1, respectively). Hindlimb muscles from mice receiving Debio 025 at 10 mg kg−1 relaxed faster, showed alteration in the stimulation frequency-dependent recruitment of muscle fibres and displayed a higher resistance to mechanical stress. Conclusions and implications: Debio 025 partially improved the structure and the function of the dystrophic mouse muscle, suggesting that therapies targeting the mPTP may be helpful to DMD patients. British Journal of Pharmacology (2008) 155, 574–584; doi:10.1038/bjp.2008.285; published online 21 July 2008

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.

The introduction of highly active antiretroviral therapy has led to significant changes in disease progression and mortality of human immunodeficiency virus (HIV) type 1 (HIV-1) infection and its sequela, AIDS. HIV-1 infection has been turned into a chronic condition that can be treated and that may be manageable over many years (90). However, a significant proportion of patients still fail to have a complete response to treatment over a prolonged time and are at risk of virological rebound, which may lead to the emergence of drug-resistant virus variants (20). The 21 anti-HIV-1 drugs used at present target only three steps in the viral replication cycle, namely, virus fusion, reverse transcription, and the proteolytic processing of viral proteins (22). An inhibitor directed against a fourth step, envelope glycoprotein binding to the CCR5 coreceptor (24, 99), and an inhibitor of a fifth step, integration of viral DNA in the cellular genome (34), were recently approved by FDA. Further inhibitors of the last two steps are in clinical evaluation (23, 86). Thus, the four virus-coded proteins, the envelope glycoprotein, reverse transcriptase (RT), protease, and integrase, are the molecular targets of anti-HIV-1 chemotherapy in the clinic at present. This limits the number of possible combinations that may be used, because cross-resistance of virus strains against drugs targeted to the same viral molecule is common (61). Therefore, novel anti-HIV drugs directed against other steps in the viral replication cycle are highly needed. Cyclophilin A (CypA) was established more than a decade ago to be a valid target in anti-HIV-1 chemotherapy (69, 70). The cellular protein CypA fulfills an essential function early in the HIV-1 replication cycle. It was found to bind specifically to the HIV-1 Gag polyprotein (48). A defined amino acid sequence around G89 and P90 of capsid protein p24 (CA) was identified as the binding site for CypA (17, 31). The affinity of CypA for CA promotes the incorporation of CypA into the virion particles during assembly (10, 30, 87). Experimental evidence indicates that the CypA-CA interaction is essential for HIV-1 replication; inhibition of this interaction impairs HIV-1 replication in human cells (10, 37, 81, 82). The step in the viral replication cycle where CypA is involved was demonstrated to be an event after penetration of the virus particle and before integration of the double-stranded viral DNA into the cellular genome (13, 51, 82). CypA is a member of the immunophilin class of proteins. These ubiquitous cellular proteins possess cis-trans prolyl isomerase (PPIase) activities (27) and are assumed to be involved in protein folding and to function as chaperones in intracellular transport (78). Cyclophilins are also known to be the intracellular receptor molecules for cyclosporines (35), a class of cyclic undecapeptides produced by Trichoderma polysporum (25, 71). Binding of cyclosporines to cyclophilins leads to the blockade of the isomerase activity. The most prominent representative of this class of compounds is cyclosporine (Cs), a potent inhibitor of T-cell activation widely used in the clinic as an immunosuppressant in organ transplantation (11). When Cs is bound to CypA it forms a ternary complex with calcineurin. This binding inhibits the phosphatase activity of calcineurin, which is crucial for signal transduction in the activation cascade of T cells. Inhibition of calcineurin function is thus the molecular basis of the immunosuppressive action of Cs (8). The structure of Cs bound to its ligands has been elucidated: two separate domains in the undecapeptide Cs that are involved in binding to CypA and calcineurin, respectively, can be distinguished (40, 43, 66, 94, 98). The immunosuppressive capacity of Cs can therefore be separated from its affinity to CypA by chemical modification. The anti-HIV-1 activity of Cs was first reported in 1988 (91). Evaluation of this drug and many derivatives for inhibition of HIV-1 replication revealed that nonimmunosuppressive Cs analogs had anti-HIV-1 activities equal to or even superior to those of immunosuppressive analogs (7, 10). A clear correlation was evident, however, between their antiviral activities and the ability of cyclosporines to bind to their cellular receptor protein, CypA. Modification of amino acids in the calcineurin binding domain of Cs, in particular, substitution of the undecapeptide at position 4, was shown to abolish the formation of the ternary complex with calcineurin and, thus, led to the loss of its immunosuppressive capacity (62, 96, 105). Binding to CypA and anti-HIV-1 activity were not impaired by such modifications. (Methyl Ile4)cyclosporine (NIM811), a nonimmunosuppressive cyclosporine with a high affinity for binding to CypA and also potent and selective anti-HIV-1 activity, was characterized in more detail (68). Mode-of-action studies revealed that NIM811 inhibited the CypA-CA interaction in a dose-dependent manner (10, 87) and that an early step in the replication cycle of HIV-1 was impaired in the presence of the compound (10, 51, 82). NIM811 was produced by fermentation of the fungus Tolypocladium niveum, followed by extraction and purification (89). Chemical derivatization procedures for the production of a large variety of Cs derivatives are well established. A large amount of knowledge exists about the structure-activity relationships regarding immunosuppressive capacity, CypA binding, and anti-HIV-1 activity (7, 10, 41, 96, 105). We set out to synthesize a series of novel cyclosporines using Cs as a starting material. The goal was to produce by chemical derivatization nonimmunosuppressive cyclosporines with increased affinities for cyclophilin, which was expected to yield derivatives with improved anti-HIV-1 activities. The most promising compound in this series of derivatives turned out to be (d-MeAla3-EtVal4)cyclosporine (Debio-025; where Me and Et are methyl and ethyl, respectively) (97). The in vitro pharmacological profile of this novel CypA-blocking compound and, in particular, its anti-HIV-1 potential are described in this report.

International audience; Duchenne muscular dystrophy (DMD) is a lethal disease caused by the lack of the cytoskeletal protein dystrophin. Altered calcium homoeostasis and increased calcium concentrations in dystrophic fibres may be responsible for the degeneration of muscle occurring in DMD. In the present study, we used subsarcolemmal- and mitochondrial-targeted aequorin to study the effect of the antiapoptotic Bcl-2 protein overexpression on carbachol-induced near-plasma membrane and mitochondrial calcium responses in myotubes derived from control C57 and dystrophic (mdx) mice. We show that Bcl-2 overexpression decreases subsarcolemmal and mitochondrial calcium overload that occurs during activation of nicotinic acetylcholine receptors in dystrophic myotubes. Moreover, our results suggest that overexpressed Bcl-2 protein may prevent near-plasma membrane and mitochondrial calcium overload by inhibiting IP3Rs (inositol 1,4,5-trisphosphate receptors), which we have shown previously to be involved in abnormal calcium homoeostasis in dystrophic myotubes. Most likely as a consequence, the inhibition of IP3R function by Bcl-2 also inhibits calcium-dependent apoptosis in these cells.

Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.

Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-l-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.

The chemical synthesis, conformational analysis and receptor binding studies of novel constrained cyclosporin A (CsA) analogues are described. The selective insertion of pseudo-proline (PsiPro) systems featuring different 2-C-substituents at the oxazolidine ring exerts dramatic effects upon the backbone conformation as demonstrated by NMR analysis. It is shown that the insertion of a Psi(MeMe)pro at position 5 (Thr(5)CsA) maintains binding to cyclophilin A as well as to calcineurin and shows a 5-6 cis amide bond with all remaining amide bonds trans. The elaborated synthetic routes for generating PsiPro containing Cs derivatives pave the way for extended structure-activity relationship studies aiming at the design of potential pharmacologically active compounds with a selective activity profile. (C) 2003 Elsevier Science Ltd. All rights reserved.

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the beta1 integrin family regulate myoblast fusion. beta1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of beta1-deficient myoblasts, suggesting that beta1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, beta1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of beta1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.

The modulation of glucose uptake by cytosolic calcium and the role of insulin on calcium homeostasis in insulin-target cells are incompletely understood and results are contradictory. To address this issue, we used the C2C12 murine skeletal muscle cell line model and examined the influence of caffeine and 4-chloro-m-cresol, two ryanodine receptor agonists known to mobilize intracellular calcium stores and increase cytosolic free calcium concentration. We followed 45 calcium efflux, a validated indicator of cytosolic calcium concentration, and 3-O-methyl-[1- 3 H]-D-glucose uptake in parallel. We also determined if insulin incubation affected 45 calcium influx rate. A 30-min treatment by 1 μM insulin highly significantly increased 45 calcium efflux by 8.5% (P = 0.0014), despite a significant reduction of 45 Ca 2+ influx already measurable after 20 and 30 min of insulin stimulation (-16.6%, P = 0.0119 and -21.3%, P = 0.0047, respectively). Caffeine (1-20 mM) and 4-chloro-m-cresol (0.05-10 mM) concentration-dependently increased 45 calcium efflux, the latter being more potent and efficacious. These agents, in a concentration-dependent manner, inhibited both basal and, more potently, insulin-stimulated glucose uptake. This resulted in a negative correlation of glucose uptake and 45 calcium efflux (r > 0 95, P < 0.001). This effect was ∼5 times greater for caffeine than for 4-chloro-m-cresol, suggesting a calcium-independent part of the glucose uptake inhibition by caffeine. In our in vitro model of cultured muscle cells, insulin appears to prevent calcium overload by both stimulating efflux and inhibiting cell storage. This effect, taken together with the observed inhibitory, inverse relationship between 45 calcium efflux and glucose uptake, contributes to describing the complex insulin-calcium interplay involved in target cells.

1. Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). 2. The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. 3. We show that CsA (1 -- 10 microM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. 4. Using cytochrome c as substrate, we show that CsA (10 microM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. 5. CsA (10 microM) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. 6. CsA (10 microM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. 7. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. 8. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes.

The mdx mouse serves as animal model for Duchenne muscular dystrophy. Energy status in muscles of mdx mice is impaired and we have demonstrated recently that the energy precursor creatine exerts beneficial effects on mdx skeletal muscle cells in culture. Here we show that feeding a creatine-enriched diet to new-born mdx mice strongly reduced the first wave of muscle necrosis four weeks after birth. Necrosis of the fast-twitch muscle extensor digitorum longus was inhibited by 63+/-14% (P

1. The major side effects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. 2. We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg8]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors. 3. Displacement experiments using a specific antagonist of the vasopressin V1A receptor (V1AR) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V1A subtype. 4. Receptor internalization studies revealed that CsA (10(-6) M) did not significantly alter AVP receptor trafficking. 5. V1AR mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time-course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. 6. A direct effect of CsA on the V1AR promoter was investigated using VSMC transfected with a V1AR promoter-luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V1AR promoter activity. This effect was independent of the cyclophilin-calcineurin pathway. 7. Measurement of V1AR mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half-life of V1AR mRNA about 2 fold. 8. In conclusion, CsA increased the response of VSMC to AVP by upregulating V1AR expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular filtration.

The pathogenesis of Duchenne muscular dystrophy (DMD), characterised by lack of the cytoskeletal protein dystrophin, is not completely understood. An early event in the degenerative process of DMD muscle could be a rise in cytosolic calcium concentration. In order to investigate whether this leads to alterations of contractile behaviour, we studied the excitability and contractile properties of cultured myotubes from control (C57BL/10) and mdx mice, an animal model for DMD. The myotubes were stimulated electrically and their motion was recorded photometrically. No significant differences were found between control and mdx myotubes with respect to the following parameters: chronaxy and rheobase (0.33 +/- 0.03 ms and 23 +/- 4 V vs. 0.39 +/- 0.07 ms and 22 +/- 2 V for C57 and mdx myotubes, respectively), tetanisation frequency (a similar distribution pattern was found between 5 and 30 Hz), fatigue during tetanus (found in 35% of both types of myotubes) and post-tetanic contracture. In contrast, contraction and relaxation times were longer (P < 0.005) in mdx (36 +/- 2 and 142 +/- 13 ms, respectively) than in control myotubes (26 +/- 1 and 85 +/- 9 ms, respectively). Together with our earlier findings, these results suggest a decreased capacity for calcium removal in mdx cells leading, in particular, to alterations of muscle relaxation.

Cyclosporine A up-regulates angiotensin II receptors and calcium responses in human vascular smooth muscle cells. Background The most widely used immunosuppressive drug for preventing graft rejection and treating autoimmune diseases is currently cyclosporine A (CsA). However, CsA also causes vasoconstriction, which is considered to be at the origin of CsA-induced nephrotoxicity and hypertension. To evaluate the cellular basis for these side effects, we studied the influence of CsA on the regulation of the free cytosolic Ca 2+ concentration ([Ca 2+ ] c ) in cultured human vascular smooth muscle cells (SMCs). Methods SMCs were isolated from the medial layer of human aorta. [Ca 2+ ] c regulation was studied by fluorimetry with fura 2 and by measuring 45 Ca 2+ effluxes. Angiotensin II (Ang II) receptors were detected by [ 125 I]Ang II binding. Results Pretreatment of human SMCs for 24hours with CsA in its therapeutic concentration range (0.1 to 10.0 μm) had no effect on basal [Ca 2+ ] c , but increased the [Ca 2+ ] c elevation and 45 Ca 2+ efflux when cells were stimulated with Ang II. Half-maximal effects occurred at approximately 1 μm CsA. The CsA effects on [Ca 2+ ] c were accompanied by a nearly twofold increase in Ang II receptor number, whereas no change in affinity to Ang II was observed. CsA did not alter endothelin-1- or thapsigargin-induced 45 Ca 2+ efflux. Increases in both Ca 2+ responses and [ 125 I]Ang II binding were attenuated by the transcriptional inhibitor actinomycin D. The effects of CsA did not appear to be mediated by calcineurin inhibition because cyclosporine H, which is not immunosuppressive, also increased the Ang II-induced 45 Ca 2+ efflux. Conclusion These data suggest that CsA preferentially up-regulates the transcription of Ang II receptors, which very likely leads to vasoconstriction in vivo and could be at the origin of CsA-induced hypertension and nephrotoxicity in humans.

Glucocorticoids, namely alpha-methylprednisolone (PDN) and deflazacort, are the only drugs reported to have a beneficial effect on the degenerative course of Duchenne muscular dystrophy (DMD). Increased cytosolic calcium concentrations ([Ca2+]c) have been implicated as one of the pathological events responsible for the degeneration of dystrophic skeletal muscles. In previous studies, we have demonstrated that PDN treatment of both normal and dystrophic murine skeletal muscle cells was able to normalize elevated [Ca2+]c and improved myogenesis. Here we have investigated the mechanism underlying the effects of glucocorticoids on cellular Ca2+ influx into C2C12 skeletal muscle cells. Long-term incubation of C2C12 myocytes with PDN was necessary to observe a reduction of 45Ca2+ influx. PDN was most effective in inhibiting 45Ca2+ uptake when added for 4 days (at the time of fusion of myoblasts into myotubes) and to a lesser extent, when added after fusion. It was ineffective when added to C2C12 cells at the myoblast stage. Short PDN incubation times, at the time of fusion were insufficient to elicit a response. Several steroids were tested for their ability to inhibit 45Ca2+ influx in C2C12 myocytes. All four glucocorticoids examined were able to reduce Ca2+ influx, dexamethasone being the most potent (IC50 3.14+/-0.34 x 10(-8) M). Mineralocorticoids (aldosterone and 11-deoxycorticosterone) were also able to reduce Ca2+ influx. The vitamin E-derived lazaroid U-83836E and the glucocorticoid-derived lazaroid U-74389G also elicited a decrease in Ca2+ influx, but higher concentrations were necessary. Because both glucocorticoids and lazaroids display antioxidant properties, but U-83836E is devoid of glucocorticoid activity, the reduction in Ca2+ influx was suspected to be triggered via an antioxidant mechanism. To test this hypothesis, we assessed the action of several antioxidants, such as vitamin E, vitamin C, 2-tert.-butyl-4-methoxyphenol (BHA), 2,6-di-tert.-butyl-4-methyl-phenol (BHT) and nordihydroguaiaretic acid (NDGA), on 45Ca2+ influx. None of these agents had an effect on 45Ca2+ influx. In addition, several oxidants were tested (either acutely or chronically) for their ability to elicit 45Ca2+ influx in C2C12 myocytes and were found to be inactive. The involvement of the glucocorticoid receptor on the modulation of Ca2+ influx was investigated. The glucocorticoid receptor antagonist mifepristone (code name RU38486, 10(-6) M) caused a shift of two orders of magnitude of the PDN response. However, neither actinomycin D nor cycloheximide affected the response to PDN. Results with the phospholipase A2 inhibitor, manoalide, suggest that glucocorticoid-induced protein synthesis (e.g. enhanced stimulation of lipocortin) does not play a role in the reduction of calcium influx. Our results suggest that steroids elicit a decrease in calcium influx in C2C12 skeletal muscle cells. This decrease is not due to an antioxidant mechanism or to a mechanism which requires gene expression. Since mineralocorticoids and U-83836E also had similar effects, the mechanism could belong to the non-genomic effects of corticoids (e.g. membrane stabilization). The beneficial effect of glucocorticoids in DMD could be attributed to a reduction of the pathological increase in Ca2+ influx via an effect on the sarcolemma.

The immunosuppressive drug cyclosporin A (CsA) depresses neutrophil oxidative burst which may lead to an increased susceptibility to infection in transplant patients. Using specific CsA analogues we investigated the mechanism of inhibition of the oxidative burst and evaluated short and long-term effects of CsA on dimethylsulphoxide-differentiated HL-60 neutrophils. A biphasic pattern was observed: a 4 h pre-treatment with CsA (1 μM) diminished the fMLP induced [Ca2+]c rise, reactive oxygen species (ROS) production, and β-glucuronidase release by about 40%, whereas a 20 h pre-treatment increased these responses by about 1.5 fold. [MeVal4]CsA, which binds with high affinity to cyclophilin but inhibits the interaction of the CsA-cyclophilin complex with calcineurin, blocked the stimulation observed with CsA after a 20 h incubation but did not alter the CsA effects after a 4 h pre-treatment. PSC 833 (1 μM), a potent multi drug resistance transporter (MDR) inhibitor, diminished ROS production to the same extent as a 4 h CsA incubation but was ineffective after a 20 h pre-treatment. An involvement of MDR as a basis for CsA or PSC 833 action was ruled out based on the results of the calcein retention assay. [3H]CsA uptake showed that CsA and [MeVal4]CsA, but not CsH or PSC 833 were strongly taken up and retained by the cells. In conclusion, the reduction of the responses after 4 h appear to be due to a primary reduction of calcium signalling, while the enhanced responses after 20 h may be due to calcineurin inhibition. British Journal of Pharmacology (1998) 124, 1774–1780; doi:10.1038/sj.bjp.0702020

The use of the immunosuppressive drug cyclosporin A (CsA) is limited by two major side effects, nephrotoxicity and hypertension, which are caused by drug-induced local vasoconstriction. We have recently shown that CsA potentiates the contraction of isolated resistance arteries to vasoconstrictor hormones and increases the calcium response to these agents in vascular smooth muscle cells (VSMC). The goal of the present study was to investigate further the molecular mechanism(s) involved in these effects. Stimulation of VSMC with [Arg]8vasopressin (AVP) induced a concentration-dependent increase in total inositol phosphates (InsP) and cellular calcium response (as measured by 45Ca2+ efflux). Preincubation of VSMC with CsA increased both InsP formation and 45Ca2+ efflux. The potentiating effect of CsA on AVP-elicited InsP formation and 45Ca2+ efflux was inhibited by co-incubation with the protein synthesis inhibitors actinomycin D and cycloheximide, indicating that CsA acted on gene expression. Binding experiments with [3H]-AVP on VSMC showed that CsA increased the number of AVP receptors by about two fold without affecting receptor affinity. Actinomycin D completely blocked this increase. These results demonstrate for the first time that incubation of VSMC with CsA increases the expression of AVP receptors, resulting in a potentiation of InsP formation and calcium response upon stimulation with AVP. This effect of CsA is likely to occur with other vasoconstrictor hormone receptors as well and could be a key mechanism in the induction of vasoconstriction, and subsequent drug-induced nephrotoxicity and hypertension. British Journal of Pharmacology (1997) 121, 248–252; doi:10.1038/sj.bjp.0701102

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.

1. In Duchenne muscular dystrophy (DMD) dysregulation of cytosolic calcium appears to be involved in the degeneration of skeletal muscle fibres. Therefore, we have studied the regulation of the free cytosolic calcium concentration ([Ca2+]c) under specific stress conditions in cultured myotubes isolated from the hind limbs of wild-type (C57BL10) and dystrophin-deficient mutant mdx mice. [Ca2+]c in the myotubes was estimated by the use of the Ca(2+)-sensitive fluorescent dye, fura-2. 2. Resting [Ca2+]c was similar in mdx and normal myotubes (35 +/- 9 nM and 38 +/- 11 nM, respectively). However, when mdx myotubes were exposed to a high extracellular calcium concentration ([Ca2+]c) of 40 mM, the [Ca2+]c was elevated to 84 +/- 29 nM, compared to 49 +/- 7 nM in normal myotubes. 3. Lowering the osmolarity of the superfusion solution from 300 mOsm to 100 mOsm resulted also in a rise in [Ca2+]c which was about two times higher for mdx (243 +/- 65 nM) than for C57BL10 (135 +/- 37 nM). Replacing extracellular Ca2+ by EGTA (0.2 mM) prevented the rise in [Ca2+]c in both mdx and normal myotubes when exposed to the low osmolarity solution. 4. Gadolinium ion (50 microM), an inhibitor of Ca2+ entry, antagonized the rise in [Ca2+]c of myotubes superfused with 40 mM [Ca2+]c by 20-40% for both mdx and C57BL10 cells, but did not significantly reduce the rise in [Ca2+]c when the cells were exposed to the hypo-osmotic buffer (100 mOsm). 5. Incubation of the cell culture for 3-5 days from the onset of induction of myotube formation with the membrane permeable protease inhibitor, calpeptin (50 microM) abolished the rise in [Ca2+]c in mdx myotubes upon exposure to hypo-osmotic shock. 6. Treatment of the cell culture for 3-5 days with alpha-methylprednisolone (PDN, 10 microM) attenuated the rise in [Ca2+]c following hypo-osmotic stress for both normal and mdx myotubes by about 50%. 7. The results described here suggest an increased permeability of mdx myotubes to Ca2+ under specific stress conditions. The ameliorating effect of PDN on [Ca2+]c could explain, at least partly, the beneficial effect of this drug on DMD patients.

We demonstrate here that stimulated 45 Ca 2+ influx in A7r5 vascular smooth muscle cells induced either by receptor activation with [Arg] 8 vasopressin or by the SR-Ca 2+ -ATPase inhibitor thapsigargin was increased more than threefold if cells were preloaded with the intracellular calcium chelator BAPTA (1,2-bis(2-aminophenoxy)ethane- N,N,N ′, N ′-tetraacetic acid). The increased influx is probably due to an attenuation of negative feedback of Ca 2+ on its own entry accompanied by increased Ca 2+ storage capacity of BAPTA-loaded cells leading to diminished cellular Ca 2+ release. We propose that BAPTA preloading could be a useful approach to investigate receptor-induced Ca 2+ influx.

1. Increased calcium (Ca2+) influx has been incriminated as a potential pathological mechanism in the chronic skeletal muscle degeneration exhibited by Duchenne muscular dystrophy (DMD) patients. We have studied the influence of the glucocorticoid alpha-methylprednisolone (PDN), the only drug known to have a beneficial effect on the degenerative course of DMD, on Ca2+ handling in the C2 skeletal muscle cell line. 2. PDN, when added 3 days (when myoblasts start to fuse into myotubes) after cell seeding, led to a 2 to 4 fold decrease in cellular Ca2+ uptake. This decrease was independent of the extracellular Ca2+ concentration applied to cells. The effect took at least 24 h in order to become established (PDN of 10(-5) M) and took longer for lower PDN concentrations (EC50 of ca. 10(-6) M at day 5, 10(-6.5) M at day 7 and 10(-7.5) M at day 9 in culture). 3. Cellular calcium accumulation was also decreased in PDN-treated myotubes exposed to 45Ca(2+)-containing medium for 1 to 6 days. 4. No effect of PDN was seen on 45Ca2+ efflux; a decrease in the amount of 45Ca2+ released was observed due to the reduction of cellular 45Ca2+ loading. 5. PDN treatment led to an approximately 2 fold decrease in basal cytosolic Ca2+ concentration. 6. Three antioxidant drugs (lazaroids), previously shown to enhance in vitro skeletal muscle cell differentiation to the same extent as PDN, induced a similar decrease in Ca2+ influx. 7. Our results suggest that long-term incubation of C2 cells with PDN leads to a decrease of the size of the cellular Ca2+ pools and to reduced resting cytosolic Ca2+ levels. Part of the beneficial effect of PDN in DMD patients could be attributed to a reduction of Ca2+ influx and of the size of Ca2+ pools in dystrophic muscle fibres.

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.

The effects of the new inotropic agent, SDZ 218-135 [(+)-(S)-4-[3-(4-diphenyl-methyl-1-piperazinyl)-2-hydroxy-propyl]- 6-(2-hydroxyethyl)-5-methyl-1,2,4-triazolo-[1,5-a]pyrimidin-7(4H)-one], were investigated using in vitro and in vivo techniques. In isolated rat atria, SDZ 218-135 elicited a dose-dependent increase in contractile force (+50% at 10 microM), which was paralleled by an increase in functional refractory period. In anesthetized rats SDZ 218-135 enhanced left ventricular (+)dP/dtmax by 100% at 10 mg/kg without influencing heart rate, arterial blood pressure, and cardiac output. In contrast to its predecessor, DPI 201-106, cardiac relaxation remained essentially unimpaired. The positive inotropic action was also maintained in a rabbit model of depressed heart function after myocardial infarction, where SDZ 218-135 increased peak acceleration of blood in the aorta. The prolongation of the effective refractory period in rat atria suggested possible antiarrhythmic effects. Indeed, SDZ 218-135 showed a dose-dependent marked reduction in reperfusion arrhythmias after coronary artery occlusion in rats. This effect was most likely due to a Class III action, since SDZ 218-135 significantly increased action potential duration (+10% at 10 microM/l) of the isolated guinea pig papillary muscle. In conclusion, SDZ 218-135 is a novel positive inotropic agent with an interesting profile of action. It does not impair cardiac relaxation and shows antiarrhythmic effects in a model of reperfusion-induced arrhythmias. The in vivo and in vitro data are consistent with a mechanism of action via sodium channel agonism.

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx 5Cv mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A 2 that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx(5Cv) mouse, an animal model for DMD. Male mdx(5Cv) mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine(®) implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx(5Cv) mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force-frequency relationship of melatonin-treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD.