Identification and mapping of protein kinase A binding sites in the costameric protein myospryn

Recently we identified a novel target gene of MEF2A named myospryn that encodes a large, muscle-specific, costamere-restricted alpha-actinin binding protein. Myospryn belongs to the tripartite motif (TRIM) superfamily of proteins and was independently identified as a dysbindin-interacting protein. Dysbindin is associated with alpha-dystrobrevin, a component of the dystrophin-glycoprotein complex (DGC) in muscle. Apart from these initial findings little else is known regarding the potential function of myospryn in striated muscle. Here we reveal that myospryn is an anchoring protein for protein kinase A (PKA) (or AKAP) whose closest homolog is AKAP12, also known as gravin/AKAP250/SSeCKS. We demonstrate that myospryn co-localizes with RII alpha, a type II regulatory subunit of PKA, at the peripheral Z-disc/costameric region in striated muscle. Myospryn interacts with RII alpha and this scaffolding function has been evolutionarily conserved as the zebrafish ortholog also interacts with PKA. Moreover, myospryn serves as a substrate for PKA. These findings point to localized PKA signaling at the muscle costamere.     

Purification of dystrophin from skeletal muscle

Dystrophin was purified from rabbit skeletal muscle by alkaline dissociation of dystrophin-glycoprotein complex which was first prepared by derivatized lectin chromatography. Dystrophin-glycoprotein complex was isolated from digitonin-solubilized rabbit skeletal muscle membranes by a novel two-step method involving succinylated wheat germ agglutinin (sWGA) chromatography and DEAE-cellulose ion exchange chromatography. Proteins co-purifying with dystrophin were a protein triplet of Mr 59,000 and four glycoproteins of Mr 156,000, 50,000, 43,000, and 35,000, all previously identified as components of the dystrophin-glycoprotein complex. Alkaline treatment of sWGA/DEAE-purified dystrophin-glycoprotein complex resulted in complete dissociation of the dystrophin-glycoprotein complex. In order to separate dystrophin from its associated proteins, alkaline-dissociated dystrophin-glycoprotein complex was sedimented by sucrose gradient centrifugation. The residual glycoproteins which contaminated peak dystrophin-containing gradient fractions were then removed by WGA-Sepharose adsorption. The resulting protein appeared as a single band with an apparent Mr of 400,000 on overloaded Coomassie Blue-stained gels. The absence of WGA-peroxidase staining on nitrocellulose transfers of the pure protein indicated that the pure protein was devoid of contaminating glycoproteins. Antisera raised against the carboxyl terminus of human skeletal muscle dystrophin (which does not cross-react with the carboxyl terminus of the chromosome 6-encoded dystrophin-related protein) recognized the pure protein as did antisera specific for the amino terminus of human dystrophin. These data indicate that the protein isolated is indeed the intact, predominant skeletal muscle isoform product of the Duchenne muscular dystrophy gene.     

Caveolin-3 directly interacts with the C-terminal tail of beta -dystroglycan. Identification of a central WW-like domain within caveolin family members

Caveolin-3, the most recently recognized member of the caveolin gene family, is muscle-specific and is found in both cardiac and skeletal muscle, as well as smooth muscle cells. Several independent lines of evidence indicate that caveolin-3 is localized to the sarcolemma, where it associates with the dystrophin-glycoprotein complex. However, it remains unknown which component of the dystrophin complex interacts with caveolin-3. Here, we demonstrate that caveolin-3 directly interacts with beta-dystroglycan, an integral membrane component of the dystrophin complex. Our results indicate that caveolin-3 co-localizes, co-fractionates, and co-immunoprecipitates with a fusion protein containing the cytoplasmic tail of beta-dystroglycan. In addition, we show that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of beta-dystroglycan that contains a PPXY motif. As the WW domain of dystrophin recognizes the same site within beta-dystroglycan, we also demonstrate that caveolin-3 can effectively block the interaction of dystrophin with beta-dystroglycan. In this regard, interaction of caveolin-3 with beta-dystroglycan may competitively regulate the recruitment of dystrophin to the sarcolemma. We discuss the possible implications of our findings in the context of Duchenne muscular dystrophy.     

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.     

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.     

Sarcoglycan complex: a muscular supporter of dystroglycan-dystrophin interplay?

In striated muscle, the cytoskeletal protein dystrophin, the protein product of the Duchenne muscular dystrophy gene, is associated with a number of sarcolemmal glycoproteins to form a large oligomeric complex, the dystrophin-glycoprotein complex (DGC). Over the last 10 years, four of these sarcolemmal glycoproteins, alpha-, beta-, gamma- and delta-sarcoglycans, have been shown to form a distinct subcomplex, the sarcoglycan complex, in the DGC. Furthermore, the genetic defects of alpha-, beta-, gamma- and delta-sarcoglycans have been identified as the causes of four distinct forms of muscular dystrophies, which are now collectively called sarcoglycanopathy. Current studies are beginning to focus on the biological functions of the sarcoglycan complex and the molecular mechanism by which its dysfunction leads to muscle cell degeneration.     

Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment

In this report, we have developed a novel method to identify compounds that rescue the dystrophin-glycoprotein complex (DGC) in patients with Duchenne or Becker muscular dystrophy. Briefly, freshly isolated skeletal muscle biopsies (termed skeletal muscle explants) from patients with Duchenne or Becker muscular dystrophy were maintained under defined cell culture conditions for a 24-h period in the absence or presence of a specific candidate compound. Using this approach, we have demonstrated that treatment with a well-characterized proteasome inhibitor, MG-132, is sufficient to rescue the expression of dystrophin, -dystroglycan, and -sarcoglycan in skeletal muscle explants from patients with Duchenne or Becker muscular dystrophy. These data are consistent with our previous findings regarding systemic treatment with MG-132 in a dystrophin-deficient mdx mouse model (Bonuccelli G, Sotgia F, Schubert W, Park D, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, and Lisanti MP. Am J Pathol 163: 1663–1675, 2003). Our present results may have important new implications for the possible pharmacological treatment of Duchenne or Becker muscular dystrophy in humans. muscular dystrophy; membrane proteins; MG-132     

Cell and fiber-type distribution of dystrophin

Duchenne muscular dystrophy is the result of dystrophin deficiency. We have determined the cell types likely to express the pathogenic effects of this neuromuscular disease by determining the pattern of dystrophin expression in normal cells. We find that all physiological types of muscle cells express dystrophin at similar levels, and that the dystrophin content of various tissues correlates with the myogenic cell population of each tissue. The dystrophin content of brain and spinal cord, however, is found not to correlate with any type of muscle cell, and it is suggested that neurons express dystrophin. The potential involvement of striated muscle fibers, the vasculature, and the nervous system in the etiology of Duchenne muscular dystrophy makes it likely that the disease is a complex disorder of combined pathogenesis. We also find that the dystrophic chicken does not represent an animal model for dystrophin deficiency.     

Mild deficiency of dystrophin-associated proteins in Becker muscular dystrophy patients having in-frame deletions in the rod domain of dystrophin.

The dystrophin-glycoprotein complex spans the sarcolemma to provide a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in skeletal muscle. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins in the sarcolemma, thus causing the disruption of the dystrophin-glycoprotein complex and the loss of the linkage to the extracellular matrix. The resulting sarcolemmal instability is presumed to render muscle fibers susceptible to necrosis. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from patients with Becker muscular dystrophy (BMD), a milder allelic form of DMD. BMD patients having in-frame deletions in the rod domain of dystrophin showed a mild to moderate reduction in all of the dystrophin-associated proteins in the sarcolemma, but this reduction was not as severe as that in DMD patients. The reduction of the immunostaining for the dystrophin-associated proteins showed a good correlation with that for dystrophin in both intensity and distribution. Our results indicate that (1) the abnormality of the sarcolemmal glycoprotein complex, which is similar to but milder than that in DMD patients, also exists in these BMD patients and (2) the rod domain of dystrophin is not crucial for the interaction with the dystrophin-associated proteins.     

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.     

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.     

Adeno-associated Virus Serotype 8 (AAV8) Delivery of Recombinant A20 to Skeletal Muscle Reduces Pathological Activation of Nuclear Factor (NF)-κB in Muscle of mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by the absence of a functional dystrophin protein. Lack of dystrophin protein disrupts the dystrophin-glycoprotein complex causing muscle membrane instability and degeneration. One of the secondary manifestations resulting from lack of functional dystrophin in muscle tissue is an increased level of cytokines that recruit inflammatory cells, leading to chronic upregulation of the nuclear factor (NF)-κB. Negative regulators of the classical NF-κB pathway improve muscle health in the mdx mouse model for DMD. We have previously shown in vitro that a negative regulator of the NF-κB pathway, A20, plays a role in muscle regeneration. Here, we show that overexpression of A20 by using a muscle-specific promoter delivered with an adeno-associated virus serotype 8 (AAV8) vector to the mdx mouse decreases activation of the NF-κB pathway in skeletal muscle. Recombinant A20 expression resulted in a reduction in number of fibers with centrally placed nuclei and a reduction in the number of T cells infiltrating muscle transduced with the AAV8–A20 vector. Taken together, we conclude that overexpression of A20 in mdx skeletal muscle provides improved muscle health by reduction of chronic inflammation and muscle degeneration. These results suggest A20 is a potential therapeutic target to ameliorate symptoms of DMD.     

Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans.

Dystrobrevin is one of the intracellular components of the transmembrane dystrophin-glycoprotein complex (DGC). The functional role of this complex in normal and pathological situations has not yet been clearly established. Dystrobrevin disappears from the muscle membrane in Duchenne muscular dystrophy (DMD), which results from dystrophin mutations, as well as in limb girdle muscular dystrophies (LGMD), which results from mutations affecting other members of the DGC complex. These findings therefore suggest that dystrobrevin may play a pivotal role in the progression of these clinically related diseases. In this study, we used the Caenorhabditis elegans model to address the question of the relationship between dystrobrevin binding to dystrophin and dystrobrevin function. Deletions of the dystrobrevin protein were performed and the ability of the mutated forms to bind to dystrophin was tested both in vitro and in a two-hybrid assay, as well as their ability to rescue dystrobrevin (dyb-1) mutations in C. elegans. The deletions affecting the second helix of the Dyb-1 coiled-coil domain abolished the binding of dystrobrevin to dystrophin both in vitro and in the two-hybrid assay. These deletions also abolished the rescuing activity of a functional transgene in vivo. These results are consistent with a model according to which dystrobrevin must bind to dystrophin to be able to function properly.     

Protein turnover is elevated in muscle of mdx mice in vivo.

mdx mice lack the protein dystrophin, the absence of which causes Duchenne muscular dystrophy in humans. To examine how mdx mice maintain muscle mass despite dystrophin deficiency, we measured protein turnover rates in muscles of mdx and wild-type (C57BL/10) mice in vivo. At all ages studied, rates of muscle protein synthesis and degradation were higher in mdx than in C57BL/10 mice.     

Drosophila Dystrophin is required for integrity of the musculature

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. The highly conserved dystrophin gene encodes a number of protein isoforms. The Dystrophin protein is part of a large protein assembly, the Dystrophin glycoprotein complex, which stabilizes the muscle membrane during contraction and acts as a scaffold for signaling molecules. How the absence of Dystrophin results in the onset of muscular dystrophy remains unclear. Here, we have used transgenic RNA interference to examine the roles of the Drosophila Dystrophin isoforms in muscle. We previously reported that one of the Drosophila Dystrophin orthologs, the DLP2 isoform, is not required to maintain muscle integrity, but plays a role in neuromuscular homeostasis by regulating neurotransmitter release. In this report, we show that reduction of all Dystrophin isoform expression levels in the musculature does not apparently affect myogenesis or muscle attachment, but results in progressive muscle degeneration in larvae and adult flies. We find that a recently identified Dystrophin isoform, Dp117, is expressed in the musculature and is required for muscle integrity. Muscle fibers with reduced levels of Dp117 display disorganized actin-myosin filaments and the cellular hallmarks of necrosis. Our results indicate the existence of at least two possibly separate roles of dystrophin in muscle, maintaining synaptic homeostasis and preserving the structural stability of the muscle.     

Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.     

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.     

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.