Rescue of dystrophic skeletal muscle by PGC-1α involves restored expression of dystrophin-associated protein complex components and satellite cell signaling.

Duchenne muscular dystrophy is typically diagnosed in the preschool years because of locomotor defects, indicative of muscle damage. Thus, effective therapies must be able to rescue muscle from further decline. We have established that peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α) gene transfer will prevent many aspects of dystrophic pathology, likely through upregulation of utrophin and increased oxidative capacity; however, the extent to which it will rescue muscle with disease manifestations has not been determined. Our hypothesis is that gene transfer of Pgc-1α into declining muscle will reduce muscle injury compared with control muscle. To test our hypothesis, adeno-associated virus 6 (AAV6) driving expression of Pgc-1α was injected into single hind limbs of 3-wk-old mdx mice, while the contralateral limb was given a sham injection. At 6 wk of age, treated solei had 37% less muscle injury compared with sham-treated muscles (P < 0.05). Resistance to contraction-induced injury was improved 10% (P < 0.05), likely driven by the five-fold (P < 0.05) increase in utrophin protein expression and increase in dystrophin-associated complex members. Treated muscles were more resistant to fatigue, which was likely caused by the corresponding increase in oxidative markers. Pgc-1α overexpressing limbs also exhibited increased expression of genes related to muscle repair and autophagy. These data indicate that the Pgc-1α pathway remains a good therapeutic target, as it reduced muscle injury and improved function using a rescue paradigm. Further, these data also indicate that the beneficial effects of Pgc-1α gene transfer are more complex than increased utrophin expression and oxidative gene expression.

Dystrobrevin isoform expression in patients with neuromuscular disease.

OBJECTIVE: The role of dystrobrevin, a cytoplasmic component of the dystrophin-protein complex, in neuromuscular diseases has not been fully elucidated. This study evaluated the expression of dystrobrevin in patients with different neuromuscular diseases. METHODS: We compared dystrobrevin isoforms expression in patients with Duchenne and Becker Muscular Dystrophy (DMD and BMD) and patients with other neuromuscular disorders not linked to the dystrophin-associated complex. RESULTS: Both, alpha-dystrobrevin-1 and -2 isoforms are markedly reduced in the muscle of patients with dystrophinopathies irrespective of the age at the time of biopsy. Conversely, alpha-dystrobrevin-1 was preserved in Limb Girdle Muscular Dystrophies (LGMD) type 2I patients with altered glycosylation of alpha-dystroglycan and in patients with alterations of alpha-dystroglycan due to defects in extracellular matrix proteins (laminin-alpha2). CONCLUSIONS: Immunolabeling of dystrobrevin could be a useful marker in the diagnostic of neuromuscular diseases.

The roles of the dystrophin-associated glycoprotein complex at the synapse.

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. A number of Duchenne patients also present with mental retardation. The dystrophin protein is part of the highly conserved dystrophin-associated glycoprotein complex (DGC) which accumulates at the neuromuscular junction (NMJ) and at a variety of synapses in the peripheral and central nervous systems. Many years of research into the roles of the DGC in muscle have revealed its structural function in stabilizing the sarcolemma. In addition, the DGC also acts as a scaffold for various signaling pathways. Here, we discuss recent advances in understanding DGC roles in the nervous system, gained from studies in both vertebrate and invertebrate model systems. From these studies, it has become clear that the DGC is important for the maturation of neurotransmitter receptor complexes and for the regulation of neurotransmitter release at the NMJ and central synapses. Furthermore, roles for the DGC have been established in consolidation of long-term spatial and recognition memory. The challenges ahead include the integration of the behavioral and mechanistic studies and the use of this information to identify therapeutic targets.

The dystrophin complex controls bk channel localization and muscle activity in Caenorhabditis elegans.

Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle.

Mechanics of muscle injury induced by lengthening contraction.

Skeletal muscle is composed of two primary structural components, contractile myofibrils and extracellular matrix (ECM). The myofibrils adhere to the surrounding endomysium through the basal lamina, sarcolemma and dystrophin, and dystrophin associated glycoprotein (DAG). In this study, a novel shear lag type model is developed to investigate the mechanics of injury to the single muscle fiber due to lengthening contractions. A single muscle fiber is considered as a composite system with reinforced by the contractile myofibrils. The lateral linkages between myofibril and endomysium is modeled as a zero thickness coating layer, that could be injured under high interfacial shear stress. The results shows that the degree of the muscle injury is correlated to the magnitude of the passive stretch during the contraction. Dystrophic muscles are more susceptible to contraction induced injury due to lack of DAG complex in lateral linkage.

Structural and functional analysis of the sarcoglycan-sarcospan subcomplex.

Sarcospan is a component of the dystrophin-glycoprotein complex that forms a tight subcomplex with the sarcoglycans. The sarcoglycan-sarcospan subcomplex functions to stabilize alpha-dystroglycan at the plasma membrane and perturbations of this subcomplex are associated with autosomal recessive limb-girdle muscular dystrophy. In order to characterize protein interactions within this subcomplex, we first demonstrate that sarcospan forms homo-oligomers within the membrane. Experiments with a panel of site-directed mutants reveal that proper structure of the large extracellular loop is an important determinant of oligo formation. Furthermore, the intracellular N- and C-termini contribute to stability of sarcospan-mediated webs. Point mutation of each cysteine residue reveals that Cys 162 and Cys 164 within the large extracellular loop form disulfide bridges, which are critical for proper sarcospan structure. The extracellular domain of sarcospan also forms the main binding site for the sarcoglycans. We propose a model whereby sarcospan forms homo-oligomers that cluster the components of the dystrophin-glycoprotein complex within the membrane.

Age-related dystrophin-glycoprotein complex structure and function in the rat extensor digitorum longus and soleus muscle.

This study tested the hypothesis that age-related changes in the dystrophin-glycoprotein complex (DGC) may precede age-associated alterations in muscle morphology and function. Compared to those in adult (6 month) rats, extensor digitorum longus (EDL) and soleus muscle mass was decreased in old (30 month) and very old (36 month) Fischer 344/NNiaHSD x Brown Norway/BiNia rats. The amount of dystrophin, beta-dystroglycan, and alpha-sarcoglycan increased with aging in the EDL and decreased with aging in the soleus. alpha-Dystroglycan levels were increased with aging in both muscles and displayed evidence of altered glycosylation. Immunostaining for the presence of antibody infiltration and dystrophin following increased muscle stretch suggested that the aging in the soleus was characterized by diminished membrane integrity. Together, these data suggest that aging is associated with alterations in EDL and soleus DGC protein content and localization. These results may implicate the DGC as playing a role in age-associated skeletal muscle remodeling.

Role of dystrophin and utrophin for assembly and function of the dystrophin glycoprotein complex in non-muscle tissue.

The dystrophin glycoprotein complex (DGC) is a multimeric protein assembly associated with either the X-linked cytoskeletal protein dystrophin or its autosomal homologue utrophin. In striated muscle cells, the DGC links the extracellular matrix to the actin cytoskeleton and mediates three major functions: structural stability of the plasma membrane, ion homeostasis, and transmembrane signaling. Mutations affecting the DGC underlie major forms of congenital muscle dystrophies. The DGC is prominent also in the central and peripheral nervous system and in tissues with a secretory function or which form barriers between functional compartments, such as the blood-brain barrier, choroid plexus, or kidney. A considerable molecular heterogeneity arises from cell-specific expression of its constituent proteins, notably short C-terminal isoforms of dystrophin. Experimentally, the generation of mice carrying targeted gene deletions affecting the DGC has clarified the interdependence of DGC proteins for assembly of the complex and revealed its importance for brain development and regulation of the 'milieu intérieur. Here, we focus on recent studies of the DGC in brain, blood-brain barrier and choroid plexus, retina, and kidney and discuss the role of dystrophin isoforms and utrophin for assembly of the complex in these tissues.

Sparks, signals and shock absorbers: how dystrophin loss causes muscular dystrophy.

The dystrophin-glycoprotein complex (DGC) can be considered as a specialized adhesion complex, linking the extracellular matrix to the actin cytoskeleton, primarily in muscle cells. Mutations in several components of the DGC lead to its partial or total loss, resulting in various forms of muscular dystrophy. These typically manifest as progressive wasting diseases with loss of muscle integrity. Debate is ongoing about the precise function of the DGC: initially a strictly mechanical role was proposed but it has been suggested that there is aberrant calcium handling in muscular dystrophy and, more recently, changes in MAP kinase and GTPase signalling have been implicated in the aetiology of the disease. Here, we discuss new and interesting developments in these aspects of DGC function and attempt to rationalize the mechanical, calcium and signalling hypotheses to provide a unifying hypothesis of the underlying process of muscular dystrophy.

The muscular dystrophies: from genes to therapies.

The genetic basis of many muscular disorders, including many of the more common muscular dystrophies, is now known. Clinically, the recent genetic advances have improved diagnostic capabilities, but they have not yet provided clues about treatment or management. Thanks to better management strategies and therapeutic interventions, however, many patients with a muscular dystrophy are more active and are living longer. Physical therapists, therefore, are more likely to see a patient with a muscular dystrophy, so understanding these muscle disorders and their management is essential. Physical therapy offers the most promise in caring for the majority of patients with these conditions, because it is unlikely that advances in gene therapy will significantly alter their clinical treatment in the near future. This perspective covers some of the basic molecular biological advances together with the clinical manifestations of the muscular dystrophies and the latest approaches to their management.