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.     

Identification of New Dystroglycan Complexes in Skeletal Muscle

The dystroglycan complex contains the transmembrane protein β-dystroglycan and its interacting extracellular mucin-like protein α-dystroglycan. In skeletal muscle fibers, the dystroglycan complex plays an important structural role by linking the cytoskeletal protein dystrophin to laminin in the extracellular matrix. Mutations that affect any of the proteins involved in this structural axis lead to myofiber degeneration and are associated with muscular dystrophies and congenital myopathies. Because loss of dystrophin in Duchenne muscular dystrophy (DMD) leads to an almost complete loss of dystroglycan complexes at the myofiber membrane, it is generally assumed that the vast majority of dystroglycan complexes within skeletal muscle fibers interact with dystrophin. The residual dystroglycan present in dystrophin-deficient muscle is thought to be preserved by utrophin, a structural homolog of dystrophin that is up-regulated in dystrophic muscles. However, we found that dystroglycan complexes are still present at the myofiber membrane in the absence of both dystrophin and utrophin. Our data show that only a minority of dystroglycan complexes associate with dystrophin in wild type muscle. Furthermore, we provide evidence for at least three separate pools of dystroglycan complexes within myofibers that differ in composition and are differentially affected by loss of dystrophin. Our findings indicate a more complex role of dystroglycan in muscle than currently recognized and may help explain differences in disease pathology and severity among myopathies linked to mutations in DAPC members.     

Gene expression profiling studies on Caenorhabditis elegans dystrophin mutants dys-1(cx-35) and dys-1(cx18)

The Caenorhabditis elegans genome contains a single dystrophin/utrophin orthologue, dys-1. Point mutations in this gene, dys-1(cx35) and dys-1(cx18), result in truncated proteins. Such mutants offer potentially valuable worm models of human Duchenne muscular dystrophy. We have used microarrays to examine genes expressed differentially between wild-type C. elegans and dys-1 mutants. We found 106 genes (115 probe sets) to be differentially expressed when the two mutants are compared to wild-type worms, 49 of which have been assigned to six functional categories. The main categories of regulated genes in C. elegans are genes encoding intracellular signalling, cell-cell communication, cell-surface, and extracellular matrix proteins; genes in these same categories have been shown by others to be differentially expressed in muscle biopsies of muscular dystrophy patients. The C. elegans model may serve as a convenient vehicle for future genetic and chemical screens to search for new drug targets.     

Muscle regeneration in mitochondrial myopathies

Mitochondrial myopathies cover a diverse group of disorders in which ragged red and COX-negative fibers are common findings on muscle morphology. In contrast, muscle degeneration and regeneration, typically found in muscular dystrophies, are not considered characteristic features of mitochondrial myopathies. We investigated regeneration in muscle biopsies from 61 genetically well-defined patients affected by mitochondrial myopathy. Our results show that the perturbed energy metabolism in mitochondrial myopathies causes ongoing muscle regeneration in a majority of patients, and some were even affected by a dystrophic morphology. The results add to the complexity of the pathogenesis underlying mitochondrial myopathies, and expand the knowledge about the impact of energy deficiency on another aspect of muscle structure and function.     

Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer

BACKGROUND: Utrophin is a large multidomain protein that belongs to a superfamily of actin-binding proteins, which includes dystrophin, alpha-actinin, beta-spectrin, fimbrin, filamin and plectin. All the members of this family contain a common actin-binding region at their N termini and perform a wide variety of roles associated with the actin cytoskeleton. Utrophin is the autosomal homologue of dystrophin, the protein defective in the X-linked Duchenne and Becker muscular dystrophies, and upregulation of utrophin has been suggested as a potential therapy for muscular dystrophy patients. RESULTS: The structure of the actin-binding region of utrophin, consisting of two calponin-homology (CH) domains, has been solved at 3.0 A resolution. It is composed of an antiparallel dimer with each of the monomers being present in an extended dumbell shape and the two CH domains being separated by a long central helix. This extended conformation is in sharp contrast to the compact monomer structure of the N-terminal actin-binding region of fimbrin. CONCLUSIONS: The crystal structure of the actin-binding region of utrophin suggests that these actin-binding domains may be more flexible than was previously thought and that this flexibility may allow domain reorganisation and play a role in the actin-binding mechanism. Thus utrophin could possibly bind to actin in an extended conformation so that the sites previously identified as being important for actin binding may be directly involved in this interaction.     

Impacts of dystrophin and utrophin domains on actin structural dynamics: implications for therapeutic design

We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and utrophin have a paradoxical effect on actin rotational dynamics-restricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here, we have evaluated individual domains of these proteins. We found that a "mini-dystrophin," lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a "micro-utrophin," with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin's actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin-protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs.     

Duchenne muscular dystrophy and the neuromuscular junction: The utrophin link

Although the precise function of utrophin at the postsynaptic membrane of the neuromuscular junction still remains unclear, despite recent genetic ‘knockout’ experiments^(1,2), a separate study in a transgenic mouse model system for Duchenne muscular dystrophy (DMD) has nonetheless shown that overexpression of utrophin into extrasynaptic regions of muscle fibers can functionally compensate for the lack of dystrophin and alleviate the muscle pathology⁽³⁾. In this context, the next step is to identify the mechanisms presiding over expression of utrophin at the neuromuscular synapse in attempts to induce its expression throughout DMD muscle fibers. In fact, additional studies have shown that an important DNA element contained with the utrophin promoter may confer synapse-specific expression to the utrophin gene^(4,5). Identification of the events culminating in the transaction of the utrophin gene within synaptic myonuclei should provide important cues for the development of an effective therapeutic strategy for DMD.     

Metabolic profiles of dystrophin and utrophin expression in mouse models of Duchenne muscular dystrophy

Metabolic profiles from ¹H nuclear magnetic resonance spectroscopy have been used to describe both one and two protein systems in four mouse models related to Duchenne muscular dystrophy using the pattern recognition technique partial least squares. Robust statistical models were built for extracts and intact cardiac tissue, distinguishing mice according to expression of dystrophin. Using metabolic profiles of diaphragm, models were built describing dystrophin and utrophin, a dystrophin related protein, expression. Increased utrophin expression counteracted some of the deficits associated with dystrophic tissue. This suggests the method may be ideal for following treatment regimes such as gene therapy.     

Absence of utrophin in intercalated discs of human cardiac muscle

Utrophin is the autosomal homologue of dystrophin. In normal skeletal muscle it is localised only to neuromuscular and myotendinous junctions, nerves and vascular tissue. In Xp21 muscular dystrophies, utrophin is also detected on the sarcolemma of skeletal and cardiac muscle, while dystrophin is absent or reduced. In normal cardiac muscle, some reports have demonstrated utrophin at intercalated discs and T-tubules. We have re-examined the distribution of utrophin in normal human cardiac muscle using a panel of eight monoclonal antibodies against different epitopes in N- and C-terminal domains. In contrast to previous studies, utrophin was not detected at the intercalated discs or T-tubules, although labelling of blood vessels was strong. We conclude that the primary location of utrophin in normal heart is in the vascular system. In addition, our results show that the utrophin on cardiac blood vessels is full length, similar to that of skeletal muscle blood vessels.     

The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries

Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.     

The NO way to increase muscular utrophin expression?

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder that results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to the search for a treatment is to compensate for dystrophin loss by utrophin, another cytoskeletal protein. During development, in normal as in dystrophic embryos, utrophin is found at the membrane surface of immature skeletal fibres and is progressively replaced by dystrophin. Thus, it is possible to consider utrophin as a 'foetal homologue' of dystrophin. In a previous work, we studied the effect of L-arginine, the substrate of nitric oxide synthetase (NOS), on utrophin expression at the muscle membrane. Using a novel antibody, we confirm here that the immunocytochemical staining was indeed due to an increase in utrophin at the sarcolemma. The result is observed not only on mdx (an animal model of DMD) myotubes in culture but also in mdx mice treated with L-arginine. In addition, we show here the utrophin increase in muscle extracts of mdx mice treated with L-arginine, after electrophoretic separation and western-blotting using this novel antibody, and thus extending the electrophoretic results previously obtained on myotube cultures to muscles of treated mice.