Subproteomics analysis of Ca+-binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle.

Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca(2+) buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca(2+) reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols, we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting, mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye 'Stains-All' was performed in order to evaluate the fate of major Ca(2+)-binding proteins in dystrophin-deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres, our analysis showed that the expression of key Ca(2+)-binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca(2+)-shuttle element, sarcalumenin. In contrast, the 'Stains-All'-positive protein spot, representing the cytosolic Ca(2+)-binding component, calmodulin, was not changed in dystrophin-deficient fibres. The reduced 2D 'Stains-All' pattern of luminal Ca(2+)-binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca(2+) buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca(2+)-binding proteins, including calsequestrin.     

Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle.

Although the reduction in dystrophin-associated glycoproteins is the primary pathophysiological consequence of the deficiency in dystrophin, little is known about the secondary abnormalities leading to x-linked muscular dystrophy. As abnormal Ca(2+) handling may be involved in myonecrosis, we investigated the fate of key Ca(2+) regulatory membrane proteins in dystrophic mdx skeletal muscle membranes. Whereas the expression of the ryanodine receptor, the dihydropyridine receptor, the Ca(2+)-ATPase, and calsequestrin was not affected, a drastic decline in calsequestrin-like proteins of 150-220 kDa was observed in dystrophic microsomes using one-dimensional immunoblotting, two-dimensional immunoblotting with isoelectric focusing, diagonal two-dimensional blotting technique, and immunoprecipitation. In analogy, overall Ca(2+) binding was reduced in the sarcoplasmic reticulum of dystrophic muscle. The reduction in Ca(2+) binding proteins might be directly involved in triggering impaired Ca(2+) sequestration within the lumen of the sarcoplasmic reticulum. Thus disturbed sarcolemmal Ca(2+) fluxes seem to influence overall Ca(2+) homeostasis, resulting in distinct changes in the expression profile of a subset of Ca(2+) handling proteins, which might be an important factor in the progressive functional decline of dystrophic muscle fibers.     

Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient muscle

The cytosolic Ca2+ -binding protein regucalcin is involved in intracellular signaling and present in high abundance in the liver. Here, we could show by comparative mass spectrometry-based proteomics screening of normal versus dystrophic fibres that regucalcin of 33.9 kDa and pI5.2 also exists in diaphragm muscle. Since the expression of sarcolemmal Ca2+ -leak channels and luminal Ca2+ -binding elements is altered in dystrophin-deficient muscle, we initiated this study in order to determine whether additional soluble muscle proteins involved in Ca2+ -handling are affected in muscular dystrophy. Following separation by two-dimensional gel electrophoresis, the spot pattern of the normal versus the mdx diaphragm muscle proteome was evaluated by densitometry. The expression levels of 20 major protein spots were shown to change and their identity determined by mass spectrometry. A 2-fold reduction of regucalcin in mdx diaphragm, as well as in dystrophic limb muscle and heart, was confirmed by immunoblotting in both young and aged mdx mice. The results from our proteomics analysis of dystrophic diaphragm support the concept that abnormal Ca2+ -handling is involved in x-linked muscular dystrophy. The reduction in key Ca2+ -handling proteins may result in an insufficient maintenance of Ca2+ -homeostasis and an abnormal regulation of Ca2+ -dependent enzymes resulting in disturbed intracellular signaling mechanisms in dystrophinopathies.     

Drastic reduction in the luminal Ca2+ -binding proteins calsequestrin and sarcalumenin in dystrophin-deficient cardiac muscle

Luminal Ca2+ -binding proteins play a central role in mediating between Ca2+ -uptake and Ca2+ -release during the excitation-contraction-relaxation cycle in muscle fibres. In the most commonly inherited neuromuscular disorder, Duchenne muscular dystrophy (DMD), the reduced expression of key Ca2+ -binding proteins causes abnormal Ca2+ -buffering in the sarcoplasmic reticulum (SR) of skeletal muscle. The heart is also affected in dystrophinopathies, as manifested by the pathological replacement of cardiac fibres by connective and fatty tissue. We therefore investigated whether similar changes occur in the abundance of luminal Ca2+ -regulatory elements in dystrophin-deficient cardiac fibres. Two-dimensional immunoblotting of total cardiac extracts was employed to unequivocally determine potential changes in the expression levels of SR components. Interestingly, the expression of the histidine-rich Ca2+ -binding protein was increased in the dystrophic heart. In contrast, the major Ca2+ -reservoir protein of the terminal cisternae, calsequestrin (CSQ), and the Ca2+ -shuttle and ion-binding protein of the longitudinal tubules, sarcalumenin, were drastically reduced in cardiac mdx fibres. This result agrees with the recently reported decrease in the Ca2+ -release channel and Ca2+ -ATPase in the mdx heart. Abnormal Ca2+ -handling appears to play a major role in the molecular pathogenesis of the cardiac involvement in X-linked muscular dystrophy.     

Depolarization-induced contraction and SR function in mechanically skinned muscle fibers from dystrophic mdx mice

Dystrophin is absent in muscle fibers of patients with Duchenne muscular dystrophy (DMD) and in muscle fibers from the mdx mouse, an animal model of DMD. Disrupted excitation-contraction (E-C) coupling has been postulated to be a functional consequence of the lack of dystrophin, although the evidence for this is not entirely clear. We used mechanically skinned fibers (with a sealed transverse tubular system) prepared from fast extensor digitorum longus muscles of wild-type control and dystrophic mdx mice to test the hypothesis that dystrophin deficiency would affect the depolarization-induced contractile response (DICR) and sarcoplasmic reticulum (SR) function. DICR was similar in muscle fibers from mdx and control mice, indicating normal voltage regulation of Ca2+ release. Nevertheless, rundown of DICR (<50% of initial) was reached more rapidly in fibers from mdx than control mice [control: 32 +/- 5 depolarizations (n = 14 fibers) vs. mdx: 18 +/- 1 depolarizations (n = 7) before rundown, P < 0.05]. The repriming rate for DICRs was decreased in fibers from mdx mice, with lower submaximal DICR observed after 5, 10, and 20 s of repriming compared with fibers from control mice (P < 0.05). SR Ca2+ reloading was not different in fibers from control and mdx mice, and no difference was observed in SR Ca2+ leak. Caffeine (2-7 mM)-induced contraction was diminished in fibers from mdx mice compared with control (P < 0.05), indicating depressed SR Ca2+ release channel activity. Our findings indicate that fast fibers from mdx mice exhibit some impairment in the events mediating E-C coupling and SR Ca2+ release channel activity.     

Decreased mAKAP, ryanodine receptor, and SERCA2a gene expression in mdx hearts

Duchenne muscular dystrophy (DMD) is a common genetic disease resulting from mutations in the dystrophin gene. The lack of dystrophin function as a cytoskeletal protein leads to abnormal intracellular Ca(2+) homeostasis, the actual source and functional consequences of which remain obscure. The mdx mouse, a mouse model of DMD, revealed alterations in contractile properties that are likely due to defective Ca(2+) handling. However, the exact mechanisms of the Ca(2+) handling defect are unclear. We performed suppressive subtractive hybridization to isolate differentially expressed genes between 5-month-old mdx and control mice. We observed a decrease in muscle A-kinase anchoring protein (mAKAP) in the mdx hearts. We noticed not only down-regulation of mAKAP mRNA but also decreased mRNA level of the molecules involved in Ca(2+) handling and excitation-contraction (E-C) coupling in the sarcoplasmic reticulum (SR), the cardiac ryanodine receptor, and the sarcoplasmic reticulum Ca(2+) ATPase. Therefore, dystrophin deficiency may cause an impairment of SR Ca(2+) homeostasis and E-C coupling in mdx hearts, in part, by decreased gene expression of molecules involved in SR Ca(2+) handling.     

Involvement of inositol 1,4,5-trisphosphate in nicotinic calcium responses in dystrophic myotubes assessed by near-plasma membrane calcium measurement

In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.     

Sarcoplasmic reticulum Ca2+ permeation explored from the lumen side in mdx muscle fibers under voltage control

Under resting conditions, external Ca(2+) is known to enter skeletal muscle cells, whereas Ca(2+) stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca(2+) entry and in the SR Ca(2+) leak is still a matter of debate, but several lines of evidence suggest that these Ca(2+) fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca(2+) dye Fura2, we first demonstrated that the rate of Ca(2+) increase in response to cyclopiazonic acid (CPA)-induced inhibition of SR Ca(2+)-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca(2+) leak. However, removal of external Ca(2+) reduced the rate of CPA-induced Ca(2+) increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca(2+) influx in mdx fibers. Fibers were then loaded with the low-affinity Ca(2+) dye Fluo5N-AM to measure intraluminal SR Ca(2+) changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca(2+) changes. Voltage dependence and magnitude of depolarization-induced SR Ca(2+) depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca(2+) reuptake activity in mdx fibers. Overall, CPA-induced SR Ca(2+) leak at -80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca(2+) in control fibers. The elevated passive SR Ca(2+) leak may contribute to alteration of Ca(2+) homeostasis in mdx muscle.     

Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse

Duchenne muscular dystrophy is the most frequent neuromuscular disorder of childhood. Although this x-linked muscle disease is extremely progressive, not all subtypes of skeletal muscles are affected in the same way. While extremities and trunk muscles are drastically weakened, extraocular muscles are usually spared in Duchenne patients. In order to determine the global protein expression pattern in these naturally protected muscles we have performed a comparative proteomic study of the established mdx mouse model of x-linked muscular dystrophy. Fluorescence difference in-gel electrophoretic analysis of 9-week-old dystrophin-deficient versus age-matched normal extraocular muscle, using a pH 4-7 gel range, identified out of 1088 recognized protein spots a moderate expression change in only seven protein species. Desmin, apolipoprotein A-I binding protein and perilipin-3 were found to be increased and gelsolin, gephyrin, transaldolase, and acyl-CoA dehydrogenase were shown to be decreased in mdx extraocular muscles. Immunoblotting revealed a drastic up-regulation of utrophin, comparable levels of β-dystroglycan and key Ca²⁺-regulatory elements, and an elevated concentration of small stress proteins in mdx extraocular muscles. This suggests that despite the lack of dystrophin only a limited number of cellular systems are perturbed in mdx extraocular muscles, probably due to the substitution of dystrophin by its autosomal homolog. Utrophin appears to prevent the loss of dystrophin-associated proteins and Ca²⁺-handling elements in extraocular muscle tissue. Interestingly, the adaptive mechanisms that cause the sparing of extraocular fibers seem to be closely linked to an enhanced cellular stress response.     

Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene

Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extarcellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex.     

Just say NO to muscle degeneration?

The devastating muscle degeneration characteristic of Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. The dystrophin complex has two functions: a structural role in maintaining sarcolemmal integrity during contraction and a scaffolding function that recruits signaling proteins such as neuronal nitric oxide synthase to the membrane. New studies indicate that transgenic restoration of nitric oxide (NO) production in the mdx dystrophic mouse improves muscle pathology. Although NO-mediated killing of inflammatory cells might be involved, other mechanisms are also possible. These results point to the therapeutic potential of manipulating the signaling activity of the dystophin complex as a way to ameliorate the progression of muscle degeneration.     

Menstrual blood-derived cells confer human dystrophin expression in the murine model of Duchenne muscular dystrophy via cell fusion and myogenic transdifferentiation

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.     

Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma

mAbs specific for protein components of the surface membrane of rabbit skeletal muscle have been used as markers in the isolation and characterization of skeletal muscle sarcolemma membranes. Highly purified sarcolemma membranes from rabbit skeletal muscle were isolated from a crude surface membrane preparation by wheat germ agglutination. Immunoblot analysis of subcellular fractions from skeletal muscle revealed that dystrophin and its associated glycoproteins of 156 and 50 kD are greatly enriched in purified sarcolemma vesicles. The purified sarcolemma was also enriched in novel sarcolemma markers (SL45, SL/TS230) and Na+/K(+)-ATPase, whereas t-tubule markers (alpha 1 and alpha 2 subunits of dihydropyridine receptor, TS28) and sarcoplasmic reticulum markers (Ca2(+)-ATPase, ryanodine receptor) were greatly diminished in this preparation. Analysis of isolated sarcolemma by SDS-PAGE and densitometric scanning demonstrated that dystrophin made up 2% of the total protein in the rabbit sarcolemma preparation. Therefore, our results demonstrate that although dystrophin is a minor muscle protein it is a major constituent of the sarcolemma membrane in skeletal muscle. Thus the absence of dystrophin in Duchenne muscular dystrophy may result in a major disruption of the cytoskeletal network underlying the sarcolemma in dystrophic muscle.     

Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.     

Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice

Dystrophin, the protein product of the human Duchenne muscular dystrophy gene, exists in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kD and a protein of 59 kD. Here, we investigated the relative abundance of each of the components of the dystrophin-glycoprotein complex in skeletal muscle from normal and mdx mice, which are missing dystrophin. Immunoblot analysis using total muscle membranes from control and mdx mice of ages 1 d to 30 wk found that all of the dystrophin-associated proteins were greatly reduced (80-90%) in mdx mouse skeletal muscle. The specificity of the loss of the dystrophin-associated glycoproteins was demonstrated by the finding that the major glycoprotein composition of skeletal muscle membranes from normal and mdx mice was identical. Furthermore, skeletal muscle membranes from the dystrophic dy/dy mouse exhibited a normal density of dystrophin and dystrophin-associated proteins. Immunofluorescence microscopy confirmed the results from the immunoblot analysis and showed a drastically reduced density of dystrophin-associated proteins in mdx muscle cryosections compared with normal and dy/dy mouse muscle. Therefore, our results demonstrate that all of the dystrophin-associated proteins are significantly reduced in mdx skeletal muscle and suggest that the loss of dystrophin-associated proteins is due to the absence of dystrophin and not due to secondary effects of muscle fiber degradation.     

Altered activity of signaling pathways in diaphragm and tibialis anterior muscle of dystrophic mice

Duchenne muscular dystrophy is a musculoskeletal disease caused by mutations in the dystrophin gene. The purpose of this study was to use the mouse model of muscular dystrophy (mdx) to determine if the progression of the dystrophic phenotype in the diaphragm (costal) versus limb skeletal muscle (tibialis anterior) is associated with specific changes in extracellular regulated kinase (ERK1/2), p70 S6 kinase (p70(S6k)), or p38 signaling pathways. The studies detected that consistent with an earlier dystrophic phenotype, phosphorylation of p70(S6k) is elevated by 40% in the diaphragm with no change in limb muscle. In addition, phosphorylation of p38 kinase was decreased by 33% in the mdx diaphragm muscle. Levels of ERK1/2 as well as phosphorylation states were elevated in the diaphragm and limb muscle of mdx mice compared with age-matched control muscles. These results indicate that distinct signaling pathways are differentially activated in skeletal muscle of mdx mice. The specificity of these responses, particularly in the diaphragm, provides insight for potential targets for blunting the progression of the muscular dystrophy phenotype.     

High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.     

Immunological hurdles in the path to gene therapy for Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD), an X-linked lethal muscle-wasting disease, have abnormal expression of the protein dystrophin within their muscle fibres. In the mdx mouse model of this condition, both germline and neonatal somatic gene transfers of dystrophin cDNAs have demonstrated the potential of gene therapy in treating DMD. However, in many DMD patients, there appears to be no dystrophin expression when muscle biopsies are immunostained or western blots are performed. This raises the possibility that the expression of dystrophin following gene transfer might trigger a destructive immune response against this 'neoantigen'. Immune responses can also be generated against the gene transfer vector used to transfect the dystrophic muscle, and the combined immune response could further damage the already inflamed muscle. These problems are now beginning to be investigated in immunocompetent mdx mice. Although much work remains to be done, there are promising indications that these immune responses might not prove as much of a concern as originally envisaged.     

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.