Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect Duchenne muscular dystrophy (DMD) is a fatal genetic disorder for which no definitive cure is available. The X-linked mutation of the dystrophin gene leads to the absence of dystrophin in skeletal muscle fibers, a biochemical defect also observed in the mdx mouse, the murine phenotype of DMD. 1 Dystrophin is a subsarcolemmal protein involved in the link between the contractile machinery and the extracellular matrix. It is generally accepted that the absence of dystrophin weakens the sarcolemma and impairs the transduction of the mechanical signal imposed by the contraction. This leads to a complex and still not fully understood network of interconnected pathogenic events responsible for progressive muscle degeneration; these events involve the increased entrance of calcium, the activation of proteases, and the occurrence of a functional ischemic state. [1][2][3][4] Recent evidence suggests that a chronic inflammatory state is a secondary reaction that strongly contributes to the progression of the pathology. A significant overexpression of inflammatory and immune response genes has been described by microarray in muscle of dystrophic subjects. 5,6 Also, activated helper and cytotoxic T cells have been found to be present in higher number in muscles of dystrophic mdx mice and to promote pathology in this phenotype. 7 According to this view, immunoSupported by Telethon-Italy (to project no. 1150) and the Association Franç ais Contre les Myopathies (as part of postdoctoral fellowships to

The highly homologous Cl À channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K D B80 lM. We also found that DIDS (4,4 0 -diisothiocyanatostilbene-2,2 0 -disulphonic acid), a generic Cl À channel blocker, inhibits CLC-Ka (K D B90 lM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs.

No abstract

Flecainide, a class IC antiarrhythmic, was shown to improve myotonia caused by sodium channel mutations in situations where the class IB antiarrhythmic drug mexiletine was less efficient. Yet little is known about molecular interactions between flecainide and human skeletal muscle sodium (hNa v 1.4) channels. Whole-cell sodium currents (I Na ) were recorded in tsA201 cells expressing wild-type (WT) and mutant hNa v 1.4 channels (R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). At a holding potential (HP) of -120 mV, flecainide use-dependently blocked WT and G1306E I Na equally but was more potent on R1448C channels. For WT, the extent of block depended on a holding voltage more negative than the activation threshold, being greater at -90 mV as compared to -120 and -180 mV. This behaviour was exacerbated by the R1448C mutation since block at -120 mV was greater than that at -180 mV. Thus flecainide can bind to inactivated sodium channels in the absence of channel opening. Nevertheless, all the channels showed the same closed-state affinity constant (K R ∼480 µM) and the same inactivated-state affinity constant (K I ∼18 µM). Simulations according to the modulated receptor hypothesis mimic the voltage-dependent block of WT and mutant channels by flecainide and mexiletine. All the results suggest similar blocking mechanisms for the two drugs. Yet, since flecainide exerts use-dependent block at lower frequency than mexiletine, it may exhibit greater benefit in all myotonic syndromes. Moreover, flecainide blocks hNa v 1.4 channel mutants with a rightward shift of availability voltage dependence more specifically than mexiletine, owing to a lower K R /K I ratio. This study offers a pharmacogenetic strategy to better address treatment in individual myotonic patients.

Background and purpose: Skeletal muscle injury by hypolipidemic drugs is not fully understood. An extensive analysis of the effect of chronic treatment with fluvastatin (5 mgkg -1 and 20 mgkg -1 ), atorvastatin (10 mgkg -1 ) and fenofibrate (60 mgkg -1 ) on rat skeletal muscle was undertaken. Experimental approach: Myoglobinemia as sign of muscle damage was measured by enzymatic assay. Histological and immunohistochemical techniques were used to estimate muscle integrity and the presence of aquaporin-4, a protein controlling water homeostasis. Electrophysiological evaluation of muscle Cl -conductance (gCl) and mechanical threshold (MT) for contraction, index of intracellular calcium homeostasis, was performed by the two-intracellular microelectrodes technique. Key results: Fluvastatin (20 mgkg -1 ) increased myoglobinemia. The lower dose of fluvastatin did not modify myoglobinemia, but reduced urinary electrolytes, suggesting direct effects on renal function. Atorvastatin also increased myoglobinemia, with slight effects on urinary parameters. No treatment caused any histological damage to muscle or modification in the number of fibres expressing aquaporin-4. Either fluvastatin (at both doses) or atorvastatin reduced sarcolemma gCl and changed MT. Both statins produced slight effects on total cholesterol, suggesting that the observed modifications occur independently of HMGCoA-reductase inhibition. Fenofibrate increased myoglobinemia and decreased muscle gCl, whereas it did not change the MT, suggesting a different mechanism of action from the statins. Conclusions and Implications This study identifies muscle gCl and MT as early targets of drugs action that may contribute to milder symptoms of myotoxicity, such as muscle cramps, while the increase of myoglobinemia is a later phenomenon.

No abstract

The role of tumour necrosis factor (TNF)-alpha or cyclo-oxygenase-2 (COX-2) eicosanoids in dystrophinopathies has been evaluated by chronically treating (4-8 weeks) adult dystrophic mdx mice with the anti-TNF-alpha etanercept (0.5 mg/kg) or the COX-2 inhibitor meloxicam (0.2 mg/kg). Throughout the treatment period the mdx mice underwent a protocol of exercise on treadmill in order to worsen the pathology progression; gastrocnemious muscles from exercised mdx mice showed an intense staining for TNF-alpha by immunohistochemistry. In vivo, etanercept, but not meloxicam, contrasted the exercise-induced forelimb force drop. Electrophysiological recordings ex vivo, showed that etanercept counteracted the decrease in chloride channel function (gCl), a functional index of myofibre damage, in both diaphragm and extensor digitorum longus (EDL) muscle, meloxicam being effective only in EDL muscle. None of the drugs ameliorated calcium homeostasis detected by electrophysiology and/or spectrofluorimetry. Etanercept, more than meloxicam, effectively reduced plasma creatine kinase (CK). Etanercept-treated muscles showed a reduction of connective tissue area and of pro-fibrotic cytokine TGF-beta1 vs. untreated ones; however, the histological profile was weakly ameliorated. In order to better evaluate the impact of etanercept treatment on histology, a 4-week treatment was performed on 2-week-old mdx mice, so to match the first spontaneous degeneration cycle. The histology profile of gastrocnemious was significantly improved with a reduction of degenerating area; however, CK levels were only slightly lower. The present results support a key role of TNF-alpha, but not of COX-2 products, in different phases of dystrophic progression. Anti-TNF-alpha drugs may be useful in combined therapies for Duchenne patients.

No abstract