Different pattern of aquaporin-4 expression in extensor digitorum longus and soleus during early development.

Aquaporin-4 (AQP4) is the neuromuscular water channel expressed at the sarcolemma of mammalian fast-twitch fibers that mediates a high water transport rate, which is important during muscle activity. Clinical interest in the neuromuscular expression of AQP4 has increased as it is associated with the protein complex formed by dystrophin, the product of the gene affected in Duchenne muscular dystrophy. The expression of AQP4 during development has not been characterized. In this study, we analyzed the expression of AQP4 in extensor digitorum longus (EDL) and soleus, a fast- and slow-twitch muscle, respectively, during the first weeks after birth. The results show that AQP4 expression in both types of skeletal muscle occurs postnatally. The time course of expression of AQP4 in the two types of muscles was also different. Whereas the expression of AQP4 protein levels in the EDL showed a progressive increase during the first month after birth, reaching levels found in adults by day 24, the levels of the protein in the soleus showed a transient peak between day 12 and day 24 and declined thereafter, an effect that may be related to the transient high number of fast motor units innervating the soleus muscle during this time. The results suggest that AQP4 expression in skeletal muscle is under neuronal influence and contribute to the understanding of the molecular events of fiber differentiation during development.

A multidisciplinary evaluation of the effectiveness of cyclosporine a in dystrophic mdx mice.

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 was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.

The alteration of calcium homeostasis in adult dystrophic mdx muscle fibers is worsened by a chronic exercise in vivo.

Chronic exercise in vivo aggravates dystrophy in mdx mice. Calcium homeostasis was evaluated ex vivo by micro-spectrofluorometry on tendon-to-tendon dissected extensor digitorum longus (EDL) muscle fibers. Resting cytosolic calcium ([Ca2+]i) and sarcolemmal permeability through Gd3+ -sensitive mechanosensitive calcium (MsCa) channel were significantly higher in mdx vs. wild-type fibers. The exercise further enhanced [Ca2+]i in mdx fibers and increased sarcolemmal permeability by activating nifedipine-sensitive leak calcium channels. The two genotypes did not differ in caffeine sensitivity and in the excitation-calcium release (ECaR) coupling mechanism by K+ depolarization. The exercise produced a similar adaptation of activation curve of ECaR and of sensitivity to caffeine. However, the inactivation of ECaR of mdx fibers did not adapt to exercise. No fiber phenotype transition occurred in exercised muscle. We provide the first evidence that an in vivo exercise worsens the impaired calcium homeostasis of dystrophic fibers, supporting the role of enhanced calcium entrance in dystrophic progression.