Dystrophin protects the sarcolemma from stresses developed during muscle contraction.

The protein dystrophin, normally found on the cytoplasmic surface of skeletal muscle cell membranes, is absent in patients with Duchenne muscular dystrophy as well as mdx (X-linked muscular dystrophy) mice. Although its primary structure has been determined, the precise functional role of dystrophin remains the subject of speculation. In the present study, we demonstrate that dystrophin-deficient muscle fibers of the mdx mouse exhibit an increased susceptibility to contraction-induced sarcolemmal rupture. The level of sarcolemmal damage is directly correlated with the magnitude of mechanical stress placed upon the membrane during contraction rather than the number of activations of the muscle. These findings strongly support the proposition that the primary function of dystrophin is to provide mechanical reinforcement to the sarcolemma and thereby protect it from the membrane stresses developed during muscle contraction. Furthermore, the methodology used in this study should prove useful in assessing the efficacy of dystrophin gene therapy in the mdx mouse.     

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.     

Prevention of dystrophin-deficient cardiomyopathy in twenty-one-month-old carrier mice by mosaic dystrophin expression or complementary dystrophin/utrophin expression

A cure for dystrophin-deficient muscular dystrophy requires treating both skeletal muscle and the heart. Whereas mosaic dystrophin expression has been shown to protect skeletal muscle, controversy exists over whether mosaic expression is protective in the heart. We have shown recently that mosaic dystrophin expression prevents stress-induced heart damage in young carrier mice. Although an interesting finding, the clinical relevance remains to be established because young dystrophin-null mdx mice do not have heart disease. On the other hand, heart failure has been reported in human carriers. To resolve this mouse/human discrepancy, we evaluated the cardiac phenotype in 21-month-old mdx, carrier, and normal mice. We found dilated cardiomyopathy in old mdx mice but not in age-matched carrier mice. All anatomical parameters and physiological assay results (ECG and closed-chest Millar catheter) were within the normal range in old carrier mice. Focal myocardial inflammation was found in a small fraction of old carrier mice, but it had no major impact on heart function. Dobutamine stress revealed a near normal hemodynamic profile except for a marginal reduction in systolic pressure in old carrier mice. Immunostaining and Western blot showed dystrophin expression in 50% cardiomyocytes in old carrier mice. Interestingly, utrophin was upregulated in dystrophin-negative heart cells in carrier mice. In summary, we have provided the first clear-cut evidence that dilated cardiomyopathy in old mdx mice was prevented by mosaic dystrophin expression or complementary dystrophin/utrophin expression. Our results raise the hope for ameliorating dystrophic cardiomyopathy through partial gene and/or cell therapy.     

Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old mdx mice. Single i.m. injections of viral vector restored dystrophin production to 25-30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent mdx mice, raising the prospects for gene therapy of muscular dystrophies.     

Metabolic and signaling alterations in dystrophin-deficient hearts precede overt cardiomyopathy

The cytoskeletal protein dystrophin has been implicated in hereditary and acquired forms of cardiomyopathy. However, much remains to be learned about the role of dystrophin in the heart. We hypothesized that the dystrophin-deficient heart displays early alterations in energy metabolism that precede overt cardiomyopathy. We evaluated the metabolic and functional phenotype of dystrophin-deficient mdx mouse hearts at 10-12 weeks, when no major histological or echocardiographic abnormalities are reported. Ex vivo working mdx heart perfusions with stable isotopes revealed a marked shift in substrate fuel selection from fatty acids to carbohydrates associated with enhanced oxygen consumption. They also unmasked in the mdx heart: (i) compromised cardiac contractile function and efficiency, (ii) reduced cellular integrity, and (iii) exacerbated alterations in mitochondrial citric acid cycle-related parameters and in nutrient signaling pathways related to Akt. The observed shift in substrate selection cannot be explained by metabolic gene remodeling. However, mdx mice hearts showed an increased expression of the atrial natriuretic factor (anf) gene, an activator of the nitric oxide (NO)/cGMP signaling pathway and marker of cardiac remodeling, and, only as the cardiomyopathy progresses (at 25 weeks of age), an increased expression of the alpha1 subunit of soluble guanylate cyclase, which is known to negatively correlate with the activity NO/cGMP pathway. Collectively, our results highlight early metabolic and signaling alterations in the dystrophin-deficient heart, which may predispose these hearts to contractile dysfunction and sarcolemmal fragility. They also suggest the presence of a "sub-clinical" defect in the NO/cGMP pathway, which in vivo, at an early age, may be compensated by enhanced anf gene expression.     

Decreased myocardial nNOS, increased iNOS and abnormal ECGs in mouse models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a devastating neuromuscular disease caused by lack of the protein, dystrophin, in skeletal muscle and heart, although the biochemical mechanism by which dystrophin loss causes muscle dysfunction is unknown. Here we show that the dystrophin-deficient mdx mouse and a mouse lacking both dystrophin and the dystrophin-related protein, utrophin (dko), have abnormal electrocardiograms (ECGs). In skeletal muscle, dystrophin is normally associated with neuronal nitric oxide synthase (nNOS) at the sarcolemma. Consequently, we have measured NOS isoform activities in hearts from control, mdx and dko mice. In control mouse hearts, eNOS and nNOS activities increased by 120% and 47%, respectively, between 2 and 6 months of age. In mdx mice, myocardial nNOS activity was decreased by 60%, 84% and 80% at 2, 6 and 12 months of age, respectively. Similarly, hearts from dko mice showed a 65% decrease in nNOS activity compared to controls at 2 months of age. Endothelial NOS (eNOS) activity was not affected by dystrophin loss, but inducible NOS (iNOS) activity was seven-fold higher than control in the mdx mouse heart by 12 months of age. We conclude that lack of dystrophin in the mdx mouse results in abnormal ECGs that are associated with decreased myocardial nNOS and increased iNOS activities.     

A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.

Adenoviral vector-mediated gene transfer offers significant potential for gene therapy of many human diseases. However, progress has been slowed by several limitations. First, the insert capacity of currently available adenoviral vectors is limited to 8 kb of foreign DNA. Second, the expression of viral proteins in infected cells is believed to trigger a cellular immune response that results in inflammation and in only transient expression of the transferred gene. We report the development of a new adenoviral vector that has all viral coding sequences removed. Thus, large inserts are accommodated and expression of all viral proteins is eliminated. The first application of this vector system carries a dual expression cassette comprising 28.2 kb of nonviral DNA that includes the full-length murine dystrophin cDNA under control of a large muscle-specific promoter and a lacZ reporter construct. Using this vector, we demonstrate independent expression of both genes in primary mdx (dystrophin-deficient) muscle cells.     

Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive and fatal genetic disorder of muscle degeneration. Patients with DMD lack expression of the protein dystrophin as a result of mutations in the X-linked dystrophin gene. The loss of dystrophin leads to severe skeletal muscle pathologies as well as cardiomyopathy, which manifests as congestive heart failure and arrhythmias. Like humans, dystrophin-deficient mice (mdx mice) show cardiac dysfunction as evidenced by a decrease in diastolic function followed by systolic dysfunction later in life. We have investigated whether sildenafil citrate (Viagra), a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction present in the mdx mice. By using echocardiography, we show that chronic sildenafil treatment reduces functional deficits in the cardiac performance of aged mdx mice, with no effect on normal cardiac function in WT controls. More importantly, when sildenafil treatment was started after cardiomyopathy had developed, the established symptoms were rapidly reversed within a few days. It is recognized that PDE5 inhibitors can have cardioprotective effects in other models of cardiac damage, but the present study reports a prevention and reversal of pathological cardiac dysfunction as measured by functional analysis in a mouse model of DMD. Overall, the data suggest that PDE5 inhibitors may be a useful treatment for the cardiomyopathy affecting patients with DMD at early and late stages of the disease.     

Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration

Plasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse, a model of Duchenne muscular dystrophy. However, sufficient long-term expression and distribution of dystrophin remain a hurdle for translating this technology into a viable treatment for Duchenne muscular dystrophy. To improve plasmid-mediated gene therapy for muscle diseases, we studied the effects of targeted plasmid integration using a phage integrase (phiC31) that can mediate the integration of suitably modified plasmids into the mammalian genome. Using a luciferase expression plasmid, we monitored plasmid gene expression noninvasively in living mice by bioluminescence imaging. Coinjection of an integrase plasmid resulted in 5- to 10-fold higher levels of sustained luciferase expression. Likewise, plasmid-mediated dystrophin expression in mdx muscle was enhanced by integration. Using a combination of dystrophin and luciferase plasmids, we analyzed the functional benefit of dystrophin expression in the dystrophic muscle. The expression of dystrophin slowed the loss of luciferase expression associated with muscle degeneration, and that protection was enhanced by targeted integration of the dystrophin plasmid. In the presence of integrase, dystrophin expression was distributed along a much greater length of individual fibers, and this was associated with increased protection against degenerative changes. These data demonstrate the importance of both the level and distribution of dystrophin expression to achieve therapeutic efficacy, and that the efficacy can be enhanced by targeted plasmid integration.     

Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery

Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.     

Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice

Duchenne muscular dystrophy (DMD) is caused by mutations in dystrophin and the subsequent disruption of the dystrophin-associated protein complex (DAPC). Utrophin is a dystrophin homolog expressed at high levels in developing muscle that is an attractive target for DMD therapy. Here we show that the extracellular matrix protein biglycan regulates utrophin expression in immature muscle and that recombinant human biglycan (rhBGN) increases utrophin expression in cultured myotubes. Systemically delivered rhBGN up-regulates utrophin at the sarcolemma and reduces muscle pathology in the mdx mouse model of DMD. RhBGN treatment also improves muscle function as judged by reduced susceptibility to eccentric contraction-induced injury. Utrophin is required for the rhBGN therapeutic effect. Several lines of evidence indicate that biglycan acts by recruiting utrophin protein to the muscle membrane. RhBGN is well tolerated in animals dosed for as long as 3 months. We propose that rhBGN could be a therapy for DMD.     

Dystrobrevin increases dystrophin's binding to the dystrophin–glycoprotein complex and provides protection during cardiac stress

Duchenne muscular dystrophy is a fatal progressive disease of both cardiac and skeletal muscle resulting from the mutations in the DMD gene and loss of the protein dystrophin. Alpha-dystrobrevin (α-DB) tightly associates with dystrophin but the significance of this interaction within cardiac myocytes is poorly understood. In the current study, the functional role of α-DB in cardiomyocytes and its implications for dystrophin function are examined. Cardiac stress testing demonstrated significant heart disease in α-DB null (adbn^−/−) mice, which displayed mortality and lesion sizes that were equivalent to those seen in dystrophin-deficient mdx mice. Despite normal expression and subcellular localization of dystrophin in the adbn^−/− heart, there is a significant decrease in the strength of dystrophin's interaction with the membrane-bound dystrophin-associated glycoprotein complex (DGC). A similar weakening of the dystrophin-membrane interface was observed in mice lacking the sarcoglycan complex. Cardiomyocytes from adbn^−/− mice were smaller and responded less to adrenergic receptor induced hypertrophy. The basal decrease in size could not be attributed to aberrant Akt activation. In addition, the organization of the microtubule network was significantly altered in adbn^−/− cardiac myocytes, while the total expression of tubulin was unchanged in adbn^−/− hearts. These studies demonstrate that α-DB is a multifunctional protein that increases dystrophin's binding to the dystrophin–glycoprotein complex, and is critical for the full functionality of dystrophin.     

Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. One of every 3,500 males suffers from DMD, yet no treatment is currently available. Genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. Adeno-associated virus (AAV) vectors, although proven superior for muscle gene transfer, are too small (5 kb) to package the 14-kb dystrophin cDNA. Here we have created a series of minidystrophin genes (<4.2 kb) under the control of a muscle-specific promoter that readily package into AAV vectors. When injected into the muscle of mdx mice (a DMD model), two of the minigenes resulted in efficient and stable expression in a majority of the myofibers, restoring the missing dystrophin and dystrophin-associated protein complexes onto the plasma membrane. More importantly, this AAV treatment ameliorated dystrophic pathology in mdx muscle and led to normal myofiber morphology, histology, and cell membrane integrity. Thus, we have defined minimal functional dystrophin units and demonstrated the effectiveness of using AAV to deliver the minigenes in vivo, offering a promising avenue for DMD gene therapy.     

Could utrophin rescue the myocardium of patients with dystrophin gene mutations?

The spontaneous up-regulation of utrophin, observed in dystrophin-deficient skeletal muscle fibers, may decrease the susceptibility of such fibers to necrosis. It has been reported that the utrophin-rescued double-mutant mdx mouse always develops a lethal cardiomyopathy. We report two patients with severe dilated cardiomyopathy due to dystrophin gene mutations: the first was a manifesting Duchenne muscular dystrophy carrier and the second a patient affected with moderate Becker muscular dystrophy. We studied their explanted heart specimen and/or endoImyocardial biopsies by immunohistochemistry and Western blot for both dystrophin and utrophin. Utrophin was found to be over-expressed in these specimens. Our results suggest that in these patients the up-regulation of utrophin in dystrophin-deficient cardiomyocytes was unable to prevent the development of life-threatening myocardial dysfunction. These findings seem to dampen the enthusiasm raised by the prospect of DMD treatment by utrophin rescue in skeletal muscle fibers, as the myocardium would still remain severely affected.     

Cardiac sodium channel Nav1.5 is regulated by a multiprotein complex composed of syntrophins and dystrophin

The cardiac sodium channel Na(v)1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Na(v)1.5 C-terminus. Pull-down experiments were performed using Na(v)1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha- and beta-syntrophin proteins. Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx(5cv) mice to study the role of this protein complex in Na(v)1.5 function. Western blot experiments revealed a 50% decrease in the Na(v)1.5 protein levels in mdx(5cv) hearts, whereas Na(v)1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx(5cv) cardiomyocytes. Finally, ECG measurements of the mdx(5cv) mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Na(v)1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.     

Consequences of disrupting the dystrophin-sarcoglycan complex in cardiac and skeletal myopathy

Mutations that disrupt the dystrophin glycoprotein complex lead to plasma membrane instability of cardiomyocytes and skeletal muscle myofibers. Instability of the plasma membrane leads to degeneration largely due to activation of a necrotic process in these disorders. In response to ongoing degeneration, skeletal muscle exhibits robust regeneration while in cardiac muscle regeneration is not obvious. The dystrophin complex is concentrated along the plasma membrane in costameric structures that correspond to the Z bands of sarcomeres, thus positioning the dystrophin complex to transmit force between the sarcomere and the plasma membrane to the extracellular matrix. Although it is apparent that this position is important for perpendicular force transmission, it is clear that the dystrophin complex also fulfills signaling roles. Nitric oxide synthase and stress-induced signaling cascades are activated to participate in protection but may also contribute to pathology.