epsilon-sarcoglycan replaces alpha-sarcoglycan in smooth muscle to form a unique dystrophin-glycoprotein complex.
(25/1292)
The sarcoglycan complex has been well characterized in striated muscle, and defects in its components are associated with muscular dystrophy and cardiomyopathy. Here, we have characterized the smooth muscle sarcoglycan complex. By examination of embryonic muscle lineages and biochemical fractionation studies, we demonstrated that epsilon-sarcoglycan is an integral component of the smooth muscle sarcoglycan complex along with beta- and delta-sarcoglycan. Analysis of genetically defined animal models for muscular dystrophy supported this conclusion. The delta-sarcoglycan-deficient cardiomyopathic hamster and mice deficient in both dystrophin and utrophin showed loss of the smooth muscle sarcoglycan complex, whereas the complex was unaffected in alpha-sarcoglycan null mice in agreement with the finding that alpha-sarcoglycan is not expressed in smooth muscle cells. In the cardiomyopathic hamster, the smooth muscle sarcoglycan complex, containing epsilon-sarcoglycan, was fully restored following intramuscular injection of recombinant delta-sarcoglycan adenovirus. Together, these results demonstrate a tissue-dependent variation in the sarcoglycan complex and show that epsilon-sarcoglycan replaces alpha-sarcoglycan as an integral component of the smooth muscle dystrophin-glycoprotein complex. Our results also suggest a molecular basis for possible differential smooth muscle dysfunction in sarcoglycan-deficient patients. (+info)
Mechanical properties of smooth muscle portal vein in normal and dystrophin-deficient (mdx) mice.
(26/1292)
Mechanical properties of the vascular smooth muscle from normal and dystrophin-deficient (mdx) mice were examined. Changes in resting and developed tensions in response to stretch were recorded in isolated portal vein. The vascular segments were elongated in 5 % increments of the 'in situ' length (Lr) up to 1.30Lr. The resting length-tension curves in male mdxmice were similar to normal mice, while a marked decrease in the slope of the curve was noted in female mdx mice. These findings were not affected by atropine, phentolamine, tetrodotoxin or [Ca2+] in the surrounding media. At Lr, the tension of isolated portal vein was characterized by spontaneous synchronized uniform force waves in normal mouse. In contrast, in mdxmouse portal veins an irregular motor pattern characterized by desynchronized force waves with a decrease of amplitude and an increase in frequency was recorded. Extension of the length of the portal vein segment did not increase the spontaneous phasic activity developed in female mdx mice although this was noted with male mdx mice and normal mice. Experiments with chemical depolarizing agents indicated that spontaneous myogenic excitation activated the great majority of vascular smooth muscle cells in normal mouse portal vein, whereas in mdx mice only a reduced number of these cells were excited suggesting that in the mdx mouse the intercellular electronic coupling is altered. In conclusion this study provides the first description of the mechanical activities of portal vein longitudinal muscle and shows that in mdx mice the motor activity is severely disrupted. (+info)
Myoblast transplantations lead to the expression of the laminin alpha 2 chain in normal and dystrophic (dy/dy) mouse muscles.
(27/1292)
Laminin-2 is part of the basement membrane of the skeletal muscle fibers. The laminin alpha 2 chain is absent or drastically reduced in a subgroup of congenital muscular dystrophy patients, and in the severely affected dystrophic dy/dy mouse. We previously reported that heterogeneous primary mouse muscle cell cultures conferred laminin alpha 2 chain expression in dy/dy mice muscles upon cell transplantation. In the present study we investigated whether pure myoblast cell lines were able to confer laminin alpha 2 chain expression in vivo. We observed that: (1) xeno-transplantation of non-immortalized human myoblast in SCID mouse muscles allows human laminin alpha 2 chain expression; (2) allotransplantation of the permanent G8 mouse myoblast cell line in dy/dy muscles allows the expression of the murine laminin alpha 2 chain; and (3) allo-transplantation of the D7 dystrophic dy/dy cell line allows the formation of new and hybrid muscle fibers in dy/dy muscle in the absence of laminin alpha 2 chain expression. We conclude that normal myoblasts are able to restore the expression of an extracellular skeletal muscle protein and that the absence of laminin-2 does not prevent transplanted muscle cells from participating in the formation of myofibers. Myoblasts are, therefore, attractive tools for further exploration of gene complementation strategies in the animal models of congenital muscular dystrophy. (+info)
Genetic polymorphism in muscle biopsies of Duchenne and Becker muscular dystrophy patients.
(28/1292)
Duchenne muscular dystrophy (DMD), with an incidence of one in 3500 male new borns, and its milder variant, Becker muscular dystrophy (BMD), are allelic X-linked recessive disorders, caused by mutations in the gene coding for dystrophin, a 427 kD cytoskeleton protein. There are no available molecular markers to differentiate these two. The purpose of this study was to study genetic polymorphism in muscular dystrophy and explore its potential in discriminating these two allelic forms of the disease. The results revealed unambiguously the presence of three transcripts : 598bp, 849bp and 1583bp long which are selectively expressed in the muscles afflicted with muscular dystrophy as compared to the normal muscle. 1583bp gene transcript was conspicuously present in the muscle tissues of both DMD and BMD patients whereas 598bp and 849bp long transcripts were exclusively present in DMD but not in BMD patients or normal human subjects. These gene transcripts had no sequence homology with dystrophin gene and these were also present in the families belonging to DMD and BMD patients. These results point to the fact that based upon the selective expression of these three gene transcripts, one could not only differentiate between DMD and BMD diseases at the molecular level, but also between normal and dystrophic muscle. Further, these findings also reveal that apart from dystrophin gene, these gene transcripts may also be responsible for the differential progression of DMD/BMD phenotype. (+info)
Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.
(29/1292)
A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation. (+info)
Different dystrophin-like complexes are expressed in neurons and glia.
(30/1292)
Duchenne muscular dystrophy is a fatal muscle disease that is often associated with cognitive impairment. Accordingly, dystrophin is found at the muscle sarcolemma and at postsynaptic sites in neurons. In muscle, dystrophin forms part of a membrane-spanning complex, the dystrophin-associated protein complex (DPC). Whereas the composition of the DPC in muscle is well documented, the existence of a similar complex in brain remains largely unknown. To determine the composition of DPC-like complexes in brain, we have examined the molecular associations and distribution of the dystrobrevins, a widely expressed family of dystrophin-associated proteins, some of which are components of the muscle DPC. beta-Dystrobrevin is found in neurons and is highly enriched in postsynaptic densities (PSDs). Furthermore, beta-dystrobrevin forms a specific complex with dystrophin and syntrophin. By contrast, alpha-dystrobrevin-1 is found in perivascular astrocytes and Bergmann glia, and is not PSD-enriched. alpha-Dystrobrevin-1 is associated with Dp71, utrophin, and syntrophin. In the brains of mice that lack dystrophin and Dp71, the dystrobrevin-syntrophin complexes are still formed, whereas in dystrophin-deficient muscle, the assembly of the DPC is disrupted. Thus, despite the similarity in primary sequence, alpha- and beta-dystrobrevin are differentially distributed in the brain where they form separate DPC-like complexes. (+info)
In vitro interactions of Caenorhabditis elegans dystrophin with dystrobrevin and syntrophin.
(31/1292)
Dystrophin, the product of the gene mutated in Duchenne muscular dystrophy (DMD) is bound by its C-terminus to a protein complex including the related protein dystrobrevin. Both proteins contain a putative coiled-coil domain consisting of two alpha-helices. It has been reported that the two proteins bind to each other by the first one of the two alpha-helices. We have revisited this question using the Caenorhabditis elegans homologs of dystrophin and dystrobrevin. In vitro interaction occurs through the more conserved second helix. We propose a new model of dystrophin interactions with associated proteins. (+info)
Strain- and age-dependent loss of sarcoglycan complex in cardiomyopathic hamster hearts and its re-expression by delta-sarcoglycan gene transfer in vivo.
(32/1292)
The delta-sarcoglycan (SG) gene is deleted in hamsters with hereditary cardiomyopathies. Immunological analyses of heart before, but not after, the progression of cardiomyopathy (CM) revealed that the BIO 14.6 strain, a model of hypertrophic CM, heterogeneously preserved alpha- and gamma-SG with loss of beta- and delta-SG. In contrast, the TO-2 strain, a model of dilated CM, did not show either SG. Furthermore, in vivo transfer of the full length delta-SG gene to TO-2 hearts expressed all four SGs. Thus, this age- and strain-dependent features suggest a more feasible setting for TO-2 than BIO 14.6 to verify both CM progression and the efficacy of gene therapy. (+info)