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.