Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes.

Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment.

Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy.

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.

Dystrobrevin deficiency at the sarcolemma of patients with muscular dystrophy.

Mutations in the genes encoding dystrophin or dystrophin-associated proteins are responsible for Duchenne muscular dystrophy or various forms of limb-girdle muscular dystrophies respectively. We have recently cloned the gene for the murine 87 kDa postsynaptic protein dystrobrevin, a dystrophin-associated protein. Anti-dystrobrevin antibodies stain the sarcolemma in normal skeletal muscle indicating that dystrobrevin co-localises with dystrophin and the dystrophin-associated protein complex. By contrast, dystrobrevin membrane staining is severely reduced in muscles of Duchenne muscular dystrophy patients, consistent with dystrobrevin being a dystrophin-associated protein. Interestingly, dystrobrevin staining at the sarcolemma is dramatically reduced in patients with limb-girdle muscular dystrophy arising from the loss of one or all of the sarcoglycan components. Normal dystrobrevin staining is observed in patients with other forms of limb-girdle muscular dystrophy where dystrophin and the rest of the dystrophin-associated protein complex are normally expressed and in other neuromuscular disorders. Our results show that dystrobrevin-deficiency is a generic feature of dystrophies linked to dystrophin and the dystrophin-associated proteins. This is the first indication that a cytoplasmic component of the dystrophin-associated protein complex may be involved in the pathogenesis of limb-girdle muscular dystrophy.

Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice.

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.

Regulation of cytosolic calcium in skeletal muscle cells of the mdx mouse under conditions of stress.

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.

Modulation by prednisolone of calcium handling in skeletal muscle cells.

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.

Antioxidant lazaroids enhance differentiation of C2 skeletal muscle cells.

We have examined the incidence of new antioxidant compounds, so-called lazaroids, on the morphological and biochemical aspects of differentiation of C2 mouse skeletal muscle cells. We show that three lazaroids (U-74006F, U-74389F and U-83836E) enhance fusion of myogenic cells when added at the day of fusion. A parallel increase in nicotinic acetylcholine receptor expression and skeletal muscle alpha-actin content was observed. This promoting effect of lazaroids on myogenesis could be related to inhibition of free radical-mediated muscle damage. Therefore, one could expect that lazaroids might be useful in the treatment of degenerating muscle diseases such as Duchenne muscular dystrophy.

Lazaroids enhance skeletal myogenesis in primary cultures of dystrophin-deficient mdx mice.

Growing evidence suggests a role for free radicals in the degeneration of dystrophin-deficient muscle (as observed in Duchenne muscular dystrophy). We therefore decided to test the action of the lazaroid antioxidant compounds on primary skeletal muscle cell cultures derived from an animal model of Duchenne muscular dystrophy, the mdx mouse. Both vitamin E-derived U-83836E and glucocorticoid-derived U-74389F enhanced myogenesis of dystrophin-deficient cultures as determined by the number of myotubes, the amount of nicotinic acetylcholine receptor, skeletal muscle alpha-actin levels and myosin light chain. U-83836E enhanced myogenesis of control congenic C57BL/10 mouse-derived muscle cultures whereas U-74389F had no detectable effect. This enhanced myogenesis was in most respects similar to the one triggered by alpha-methylprednisolone which is the only drug known to be beneficial in Duchenne muscular dystrophy.

Prednisolone enhances myogenesis and dystrophin-related protein in skeletal muscle cell cultures from mdx mouse.

The differentiation of skeletal muscle cells from mdx mice which lack dystrophin expression was examined after glucocorticoid treatment, namely alpha-methylprednisolone (PDN). Primary skeletal muscle cell cultures were established from newborn mdx, congenic C57BL/10, and allogenic BALB/C mice. We show that PDN promotes the myogenesis of both mdx- and control mice-derived cultures as determined by 1) the number of myotubes, 2) acetylcholine receptors, and 3) dystrophin and dystrophin-related protein levels. These results support the hypothesis that PDN could enhance the myogenesis of satellite cells and increase dystrophin-related protein expression in DMD treated patients.

alpha-Methylprednisolone promotes skeletal myogenesis in dystrophin-deficient and control mouse cultures.

We have examined the influence of the glucocorticoid alpha-methylprednisolone (PDN) on the morphological differentiation of skeletal muscle cells derived from dystrophin-deficient C57BL/10 mdx, congenic C57BL/10 and allogenic Balb/c newborn mice. We show that PDN enhances myogenic cell numbers in dystrophin-deficient cultures as well as in matched controls. A parallel increase in the fusion rate of myoblasts into myotubes occurs while the size of myotubes, as determined by nuclei per myotube, is slightly increased. This promoting effect of PDN on myogenesis could be related to the enhanced muscular function observed in PDN-treated Duchenne's muscular dystrophy-affected boys.

A rapid preparation of primary cultures of mouse skeletal muscle cells.

We describe a rapid and reproducible technique for establishing primary cultures of skeletal muscle cells from mouse origin. This method was aimed at avoiding extensive enzymatic proteolysis which is commonly used for preparation of primary skeletal muscle cultures. It relies on a Stomacher blender that allows a rapid and regular mechanical dissociation of muscle samples by repeated shocks. Cultures have been compared to those obtained by a modification of the method of Yaffé (1993) based on tryptic dissociation of rat muscle thighs. The time of preparation was reduced to 1 h and 15 min as compared to 4 h with the technique of Yaffé. Both cultures displayed similar morphologies and exhibited comparable myogenesis processes. Cellular yield, rate of myotube formation and myotube numbers were similar. The expression of myogenesis markers were identical as assessed by determination of acetylcholine receptor number, creatine kinase activity and level of myosin light chain.