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Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.

The glycosylation of dystroglycan is required for its function as a high-affinity laminin receptor, and loss of dystroglycan glycosylation results in congenital muscular dystrophy. The purpose of this study was to investigate the functional detects in slow- and fast-twitch muscles of glycosylation-deficient Largemyd mice. While a partial alteration in glycosylation of dystroglycan in heterozygous [Large.sup.myd/+] mice was not sufficient to alter muscle function, homozygous [Large.sup.myd/myd] mice demonstrated a marked reduction in specific force in both soleus and extensor digitorum longus (EDL) muscles. Although EDL muscles from [Large.sup.myd/myd] mice were highly susceptible to lengthening contraction-induced injury, [Large.sup.myd/myd] soleus muscles surprisingly showed no greater force deficit compared with wild-type soleus muscles even after five lengthening contractions. Despite no increased susceptibility to injury, [Large.sup.myd/myd] soleus muscles showed loss of dystroglycan glycosylation and laminin binding activity and dystrophic pathology. Interestingly, we show that soleus muscles have a markedly higher sarcolemma expression of [[beta].sub.1]-containing integrins compared with EDL and gastrocnemius muscles. Therefore, we conclude that [[beta].sub.1]-containing integrins play an important role as matrix receptors in protecting muscles containing slow-twitch fibers from contraction-induced injury in the absence of dystroglycan function, and that contraction-induced injury appears to be a separable phenotype from the dystrophic pathology of muscular dystrophy. LARGE: extracellular matrix; dystroglycan; integrin doi: 10.1152/ajpcell.00192.2010.

Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by [alpha]-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin [alpha]7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by [Large.sup.myd] muscles, which have an intact dystrophin-glycoprotein complex and lack only the laminin globular domain-binding motif on [alpha]-dystroglycan. Compromised sarcolemmal integrity is directly shown in [Large.sup.myd] muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding [alpha]-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage. dystroglycanopathy | glycosylation | integrin | membrane damage | muscular dystrophy

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin. 2,3-butanedione monoximine; gene transfer; adenovirus

Several congenital muscular dystrophies caused by defects in known or putative glycosyltransferases are commonly associated with hypoglycosylation of [alpha]-dystroglycan ([alpha]-DG) and a marked reduction of its receptor function. We have investigated changes in the processing and function of [alpha]-DG resulting from genetic manipulation of LARGE, the putative glycosyltransferase mutated both in Large[sup.myd] mice and in humans with congenital muscular dystrophy 1D (MDC1D). Here we show that overexpression of LARGE ameliorates the dystrophic phenotype of Large[sup.myd] mice and induces the synthesis of glycan-enriched [alpha]-DG with high affinity for extracellular ligands. Notably, LARGE circumvents the [alpha]-DG glycosylation defect in cells from individuals with genetically distinct types of congenital muscular dystrophy. Gene transfer of LARGE into the cells of individuals with congenital muscular dystrophies restores [alpha]-DG receptor function, whereby glycan-enriched [alpha]-DG coordinates the organization of laminin on the cell surface. Our findings indicate that modulation of LARGE expression or activity is a viable therapeutic strategy for glycosyltransferase-deficient congenital muscular dystrophies., Author(s): Rita Barresi [1]; Daniel E Michele [1]; Motoi Kanagawa [1]; Hollie A Harper [1]; Sherri A Dovico [1]; Jakob S Satz [1]; Steven A Moore [2]; Wenli Zhang [3]; [...]

The pathogenesis of muscular dystrophy is manifested via perturbation of satellite cell function, which in turn disrupts the dystroglycan (DAG1) gene and dystrophin-glycoprotein complex, and insufficient repair of skeletal muscle cells leading to muscular dystrophy. However, the self-renewing potential is not decreased during aging and during repetitive cycles of muscle de- and regeneration.

Author(s): Steven A. Moore [1]; Fumiaki Saito [2]; Jianguo Chen [3]; Daniel E. Michele [2]; Michael D. Henry [2]; Albee Messing [4]; Ronald D. Cohn [2]; Susan E. Ross-Barta [1]; [...]

Author(s): Daniel E. Michele [1]; Rita Barresi [1]; Motoi Kanagawa [1]; Fumiaki Saito [1]; Ronald D. Cohn [1]; Jakob S. Satz [1]; James Dollar [2]; Ichizo Nishino [3]; Richard I. [...]

Cardiac myocytes that have mutations in the gene for alpha-tropomyosin can produce inappropriate force during conditions of submaximal calcium levels. This indicates that these mutations can cause hypertrophic cardiomyopathy.

Plasma membrane repair is an evolutionarily conserved mechanism by which cells can seal breaches in the plasma membrane. Mutations in several proteins with putative roles in sarcolemma integrity, membrane repair, and membrane transport result in several forms of muscle disease; however, the mechanisms that are activated and responsible for sarcolemma resealing are not well understood. Using the standard assays for membrane repair, which track the uptake of FM 1‐43 dye into adult skeletal muscle fibers following laser‐induced sarcolemma disruption, we show that labeling of resting fibers by FM1‐43 prior to membrane wounding and the induced FM1‐43 dye uptake after sarcolemma wounding occurs via dynamin‐dependent endocytosis. Dysferlin‐deficient muscle fibers show elevated dye uptake following wounding, which is the basis for the assertion that membrane repair is defective in this model. Our data show that dynamin inhibition mitigates the differences in FM1‐43 dye uptake between dysferlin‐null and wild‐type muscle fibers, suggesting that elevated wound‐induced FM1‐43 uptake in dysferlin‐deficient muscle may actually be due to enhanced dynamin‐dependent endocytosis following wounding, though dynamin inhibition had no effect on dysferlin trafficking after wounding. By monitoring calcium flux after membrane wounding, we show that reversal of calcium precedes the sustained, slower increase of dynamin‐dependent FM1‐43 uptake in WT fibers, and that dysferlin‐deficient muscle fibers have persistently increased calcium after wounding, consistent with its proposed role in resealing. These data highlight a previously unappreciated role for dynamin‐dependent endocytosis in wounded skeletal muscle fibers and identify overactive dynamin‐dependent endocytosis following sarcolemma wounding as a potential mechanism or consequence of dysferlin deficiency. [ABSTRACT FROM AUTHOR]

The importance of the N-terminal domain and a portion of the mucin-like domain of alpha-dystroglycan for high-affinity laminin-receptor function are examined. The molecular recognition of dystroglycan by LARGE is the key determinant in the biosynthetic pathway for the production of mature and functional dystroglycan.