Dystrophin In Muscle Research Articles

Respiratory performance in Duchenne muscular dystrophy: Clinical manifestations and lessons from animal models.

Duchenne muscular dystrophy (DMD) is a fatal genetic neuromuscular disease. Lack of dystrophin in skeletal muscles leads to intrinsic weakness, injury, subsequent degeneration and fibrosis, decreasing contractile function. Dystropathology eventually presents in all inspiratory and expiratory muscles of breathing, severely curtailing their critical function. In people with DMD, premature death is caused by respiratory or cardiac failure. There is an urgent need to develop therapies that improve quality of life and extend life expectancy in DMD. Surprisingly, there is a dearth of information on respiratory control in animal models of DMD, and respiratory outcome measures are often limited or absent in clinical trials. Characterization of respiratory performance in murine and canine models has revealed extensive remodelling of the diaphragm, the major muscle of inspiration. However, significant compensation by extradiaphragmatic muscles of breathing is evident in early disease, contributing to preservation of peak respiratory system performance. Loss of compensation afforded by accessory muscles in advanced disease is ultimately associated with compromised respiratory performance. A new and potentially more translatable murine model of DMD, the D2.mdx mouse, has recently been developed. Respiratory performance in D2.mdx mice is yet to be characterized fully. However, based on histopathological features, D2.mdx mice might serve as useful preclinical models, facilitating the testing of new therapeutics that rescue respiratory function. This review summarizes the pathophysiological mechanisms associated with DMD both in humans and in animal models, with a focus on breathing. We consider the translational value of each model to human DMD and highlight the urgent need for comprehensive characterization of breathing in representative preclinical models to better inform human trials.

Increase in cathepsin K gene expression in Duchenne muscular dystrophy skeletal muscle.

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

On genotype-phenotype relationship of dystrophinopathies among Iranian population.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are inherited X-linked disorders resulting from alterations in the dystrophin gene. Genotype-phenotype matching studies have revealed a link between disease severity, the amount of muscle dystrophin, and the extent of mutation/deletion on the dystrophin gene. This study aimed to assess the relationship between genetic alterations in the dystrophin gene and the clinical status of patients with dystrophinopathies among the Iranian population. Methods: This cross-sectional study examined 54 patients with muscle weakness caused by abnormalities in the dystrophin gene at a hospital affiliated to Isfahan University of Medical Sciences, Isfahan, Iran, in 2021. The participants' demographic information, including age, family history of muscle dystrophies, and family history of other medical diseases as well as the type of muscular dystrophy were recorded. Furthermore, the number and region of deleted exons based on dystrophy types were also evaluated using multiplex ligation-dependent probe amplification (MLPA). The patients' gaits were also assessed as using a wheelchair, the presence of waddling gaits, or toe gaits. The patients' clinical status and the coexistence of pulmonary, bulbar, and mental conditions were also examined and compared between the two groups of dystrophinopathies. Results: In this study, 54 patients with dystrophinopathy with the mean age of 16.63 ± 12.10 years were evaluated, of whom 22 (40.7%) and 30 (55.6%) patients were classified as BMD and DMD, respectively. The most affected regions with deleted exons were exons 45-47 (n = 5) and 45-48 (n = 4) in patients with BMD, while exons 45, 48-52, 51-55, and 53 (2 cases per exon) were the most common affected exons in patients with DMD. Further analyses revealed that deletions in exons 45-47 and 51-55 were significantly associated with older and younger ages at the onset of becoming wheelchair-bound in patients with dystrophy, respectively. The hotspot range in both BMD and DMD was within exons 45-55 (n = 15 for each group); 63% of the patients had alterations on the dystrophin gene within this range [30 patients (68.18%) in the BMD group, 15 patients (53.57%) in the DMD group]. Conclusion: Exon deletion was the most common genetic alteration in patients with dystrophinopathies. No significant difference was observed between DMD and BMD regarding the number of deleted exons. Deletions in exons 45-47 and 51-55 were linked to later and earlier onset of becoming wheelchair-bound, respectively.

Extracellular Matrix Proteomics: The mdx-4cv Mouse Diaphragm as a Surrogate for Studying Myofibrosis in Dystrophinopathy.

The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the DMD gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy. Animal model research has been instrumental in the characterization of dystrophic muscles and has contributed to a better understanding of the complex pathogenesis of dystrophinopathies, the discovery of new disease biomarkers, and the testing of novel therapeutic strategies. In this article, we review how mass-spectrometry-based proteomics can be used to study changes in key components of the endomysium, perimysium, and epimysium, such as collagens, proteoglycans, matricellular proteins, and adhesion receptors. The mdx-4cv mouse diaphragm displays severe myofibrosis, making it an ideal model system for large-scale surveys of systematic alterations in the matrisome of dystrophic fibers. Novel biomarkers of myofibrosis can now be tested for their appropriateness in the preclinical and clinical setting as diagnostic, pharmacodynamic, prognostic, and/or therapeutic monitoring indicators.

Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.

Multiomic characterization of disease progression in mice lacking dystrophin.

Duchenne muscular dystrophy (DMD) is caused by genetic mutations leading to lack of dystrophin in skeletal muscle. A better understanding of how objective biomarkers for DMD vary across subjects and over time is needed to model disease progression and response to therapy more effectively, both in pre-clinical and clinical research. We present an in-depth characterization of disease progression in 3 murine models of DMD by multiomic analysis of longitudinal trajectories between 6 and 30 weeks of age. Integration of RNA-seq, mass spectrometry-based metabolomic and lipidomic data obtained in muscle and blood samples by Multi-Omics Factor Analysis (MOFA) led to the identification of 8 latent factors that explained 78.8% of the variance in the multiomic dataset. Latent factors could discriminate dystrophic and healthy mice, as well as different time-points. MOFA enabled to connect the gene expression signature in dystrophic muscles, characterized by pro-fibrotic and energy metabolism alterations, to inflammation and lipid signatures in blood. Our results show that omic observations in blood can be directly related to skeletal muscle pathology in dystrophic muscle.

Unusually severe muscular dystrophy upon in-frame deletion of the dystrophin rod domain and lack of compensation by membrane-localized utrophin

Utrophin, a dystrophin homolog, is consistently upregulated in muscles of patients with Duchenne muscular dystrophy (DMD) and is believed to partially compensate for the lack of dystrophin in dystrophic muscle. Even though several animal studies support the idea that utrophin can modulate DMD disease severity, human clinical data are scarce. We describe a patient with the largest reported in-frame deletion in the DMD gene, including exons 10-60 and thus encompassing the entire rod domain. The patient presented with an unusually early and severe progressive weakness, initially suggesting congenital muscular dystrophy. Immunostaining of his muscle biopsy showed that the mutant protein was able to localize at the sarcolemma and stabilize the dystrophin-associated complex. Strikingly, utrophin protein was absent from the sarcolemmal membrane despite the upregulation of utrophin mRNA. Our results suggest that the internally deleted and dysfunctional dystrophin lacking the entire rod domain may exert a dominant-negative effect by preventing upregulated utrophin protein from reaching the sarcolemmal membrane and thus blocking its partial rescue of muscle function. This unique case may set a lower size limit for similar constructs in potential gene therapy approaches. This work was supported by a grant from MDA USA (MDA3896) and by grant number R01AR051999 from NIAMS/NIH to C.G.B.

T Cell Responses to Dystrophin in a Natural History Study of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, but many patients have rare revertant fibers that express dystrophin. The skeletal muscle pathology of DMD patients includes immune cell infiltration and inflammatory cascades. There are several strategies to restore dystrophin in skeletal muscles of patients, including exon skipping and gene therapy. There is some evidence that dystrophin restoration leads to a reduction in immune cells, but dystrophin epitopes expressed in revertant fibers or following genome editing, cell therapy, or microdystrophin delivery after adeno-associated viral gene therapy may elicit T cell production in patients. This may affect the efficacy of the therapeutic intervention, and potentially lead to serious adverse events. To confirm and extend previous studies, we performed annual enzyme- linked immunospot interferon-gamma assays on peripheral blood mononuclear cells from 77 pediatric boys with DMD recruited into a natural history study, 69 of whom (89.6%) were treated with corticosteroids. T cell responses to dystrophin were quantified using a total of 368 peptides spanning the entire dystrophin protein, organized into nine peptide pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Six (7.8%) patients had a T cell-mediated immune response to dystrophin at at least one time point. All patients who had a positive result had been treated with corticosteroids, either prednisolone or prednisone. Our results show that ∼8% of DMD individuals in our cohort have a pre-existing T cell-mediated immune response to dystrophin, despite steroid treatment. Although these responses are relatively low level, this information should be considered a useful immunological baseline before undertaking clinical trials and future DMD studies. We further highlight the importance for a robust, reproducible standard operating procedure for collecting, storing, and shipping samples from multiple centers to minimize the number of inconclusive data.

Urine titin as a novel biomarker for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most severe form of muscular dystrophy that is caused by lack of dystrophin, a critical structural protein in skeletal muscle. DMD treatments, and quantitative biomarkers to assess the efficacy of potential treatments, are urgently needed. Previous evidence has shown that titin, a muscle cell protein, is increased in the urine of patients with DMD, suggesting its usefulness as a DMD biomarker. Here, we demonstrated that the elevated titin in urine is directly associated with the lack of dystrophin and urine titin responses to drug treatment. We performed a drug intervention study using mdx mice, a DMD mouse model. We showed that mdx mice, which lack dystrophin due to a mutation in exon 23 of the Dmd gene, have elevated urine titin. Treatment with an exon skipper that targets exon 23 rescued muscle dystrophin level and dramatically decreased urine titin in mdx mice and correlates with dystrophin expression. We also demonstrated that titin levels were significantly increased in the urine of patients with DMD. This suggests that elevated urine titin level might be a hallmark of DMD and a useful pharmacodynamic marker for therapies designed to restore dystrophin levels.

Re-examination of therapeutic management of muscular dystrophies using a vascular smooth muscle-centered approach.

In contrast to the long-standing focus on the pathophysiology of skeletal muscles in the hunt for a cure for Duchenne muscular dystrophy (DMD), we opine that the malfunctioning of dystrophin produced by vascular smooth muscle is a major contributor to the pathology of the illness. We believe that a biological response modifier glucan (BRMG), which has been shown in clinical studies of DMD to boost the expression of vascular smooth muscle dystrophin and provide anti-fibrotic and anti-inflammatory effects, may play a key role in reducing the pathogenesis of DMD. According to the evaluation of biomarkers, this BRMG, which is safe and side-effect-free, reduces the pathogenesis of DMD. We describe the possible mechanisms of action by which this BRMG helps in alleviating the symptoms of DMD by targeting smooth muscle dystrophin, in addition to its advantages over other therapeutic modalities, as well as how it can serve as a valuable adjunct to existing therapies. We suggest that using BRMG adjuncts that target smooth muscle dystrophin would be a potential therapeutic approach that prolongs the lifespan and extends the duration of ambulation from the onset of DMD. Further studies are needed to validate this hypothesis.

Systemic PPMO-mediated dystrophin expression in the Dup2 mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease that arises due to the loss of dystrophin expression, leading to progressive loss of motor and cardiorespiratory function. Four exon-skipping approaches using antisense phosphorodiamidate morpholino oligomers (PMOs) have been approved by the FDA to restore a DMD open reading frame, resulting in expression of a functional but internally deleted dystrophin protein, but in patients with single-exon duplications, exon skipping has the potential to restore full-length dystrophin expression. Cell-penetrating peptide-conjugated PMOs (PPMOs) have demonstrated enhanced cellular uptake and more efficient dystrophin restoration than unconjugated PMOs. In the present study, we demonstrate widespread PPMO-mediated dystrophin restoration in the Dup2 mouse model of exon 2 duplication, representing the most common single-exon duplication among patients with DMD. In this proof-of-concept study, a single intravenous injection of PPMO targeting the exon 2 splice acceptor site induced 45% to 68% exon 2-skipped Dmd transcripts in Dup2 skeletal muscles 15days post-injection. Muscle dystrophin restoration peaked at 77% to 87% average dystrophin-positive fibers and 41% to 51% of normal signal intensity by immunofluorescence, and 15.7% to 56.8% of normal by western blotting 15 to 30days after treatment. These findings indicate that PPMO-mediated exon skipping is a promising therapeutic strategy for muscle dystrophin restoration in the context of exon 2 duplications.

Good news for the mdx mouse community: Improved dystrophin restoration after skipping mouse dystrophin exon 23

Good news for the mdx mouse community: Improved dystrophin restoration after skipping mouse dystrophin exon 23

Abnormal Expression of Synaptic and Extrasynaptic GABAA Receptor Subunits in the Dystrophin-Deficient mdx Mouse.

Duchenne muscular dystrophy (DMD) is a neurodevelopmental disorder primarily caused by the loss of the full-length Dp427 dystrophin in both muscle and brain. The basis of the central comorbidities in DMD is unclear. Brain dystrophin plays a role in the clustering of central gamma-aminobutyric acid A receptors (GABAARs), and its loss in the mdx mouse alters the clustering of some synaptic subunits in central inhibitory synapses. However, the diversity of GABAergic alterations in this model is still fragmentary. In this study, the analysis of in vivo PET imaging of a benzodiazepine-binding site radioligand revealed that the global density of central GABAARs is unaffected in mdx compared with WT mice. In contrast, semi-quantitative immunoblots and immunofluorescence confocal imaging in tissue sections revealed complex and differential patterns of alterations of the expression levels and/or clustered distribution of a variety of synaptic and extrasynaptic GABAAR subunits in the hippocampus, cerebellum, cortex, and spinal cord. Hence, dystrophin loss not only affects the stabilization of synaptic GABAARs but also influences the subunit composition of GABAARs subtypes at both synaptic and extrasynaptic sites. This study provides new molecular outcome measures and new routes to evaluate the impact of treatments aimed at compensating alterations of the nervous system in DMD.

Dystrophin Restoration after Adeno-Associated Virus U7–Mediated Dmd Exon Skipping Is Modulated by Muscular Exercise in the Severe D2-Mdx Duchenne Muscular Dystrophy Murine Model

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by Dmd mutations, resulting in the absence of dystrophin in skeletal muscle, with a greater susceptibility to damage during contraction (exercise). In this study, we evaluated whether voluntary exercise impacts a Dmd exon skipping approach and muscle physiology in a severe DMD murine model. We intramuscularly injected an adeno-associated virus (AAV) U7 snRNA that aimed to correct Dmd reading frame in D2-mdx mice, and they voluntary ran in a wheel for 1 month. Our results suggested that voluntary running did not induce muscle fiber regeneration, considering the percentage of centronucleated fibers, Myh3 and Myh4 expression, or maximal force production, and thus possibly not compromise a gene therapy approach. With the AAV-U7 injected just before exercise initiation, we reported that voluntary running did not impact the number of viral genomes and the expression of U7 and Dmd 1 month after injection of AAV-U7, but reduced the amount of dystrophin, from 80% to 65% of the muscle cross-sectional area with dystrophin-expressing fibers. In conclusion, voluntary running did not induce muscle damage and had no drastic detrimental effect on the AAV gene therapy exon skipping approach in a severe murine DMD model. Moreover, our results suggested that exercise should be an additional element to consider in the design and conception of future therapeutic approaches for DMD.

Dystrophin-Glycoprotein Complex Behavior in Sternocleidomastoid Muscle of High- and Low-Ranking Baboons: A Possible Phylogenetic Arrangement.

The dystrophin-glycoprotein complex is a multimeric system made up of the sarcoglycan sub-complex, the sarcomplasmatic complex and the dystroglycans complex. The sarcoglycan sub-complex stabilizes the sarcolemma during muscle activity and plays a role in force transduction. This protein system is also expressed in the muscle of non-human primates such as chimpanzees and baboons, and its expression changes depending on social ranking. In fact, previous data have shown that all muscle fibers of masseter and sternocleidomastoid muscles of chimpanzees and high- ranking baboons always express sarcoglycans, while middle- and low-ranking baboons are characterized by fibers that are negative for the sarcoglycan sub-complex. Given this information, the aim of the present work was to evaluate the expression of other proteins such as laminin, beta dystroglycan and dystrophin in the sternocleidomastoid muscle of high- and low-ranking baboons. The samples were processed by immunohistochemistry; results show that in high-ranking baboons, all tested proteins were always expressed while in low-ranking baboons, fibers that were negative for sarcoglycans and beta dystroglycan have been observed. No negative fibers for laminin and dystrophin have been found in low-ranking baboons suggesting that only the transmembrane proteins of the dystrophin glycoprotein complex change in their expression and that could be correlated to a phylogenetic arrangement.

Proteomic profiling of impaired excitation-contraction coupling and abnormal calcium handling in muscular dystrophy.

The X-linked inherited neuromuscular disorder Duchenne muscular dystrophy is characterised by primary abnormalities in the membrane cytoskeletal component dystrophin. The almost complete absence of the Dp427-M isoform of dystrophin in skeletal muscles renders contractile fibres more susceptible to progressive degeneration and a leaky sarcolemma membrane. This in turn results in abnormal calcium homeostasis, enhanced proteolysis and impaired excitation-contraction coupling. Biochemical and mass spectrometry-based proteomic studies of both patient biopsy specimens and genetic animal models of dystrophinopathy have demonstrated significant changes in the concentration and/or physiological function of essential calcium-regulatory proteins in dystrophin-lacking voluntary muscles. Abnormalities include dystrophinopathy-associated changes in voltage sensing receptors, calcium release channels, calcium pumps and calcium binding proteins. This review article provides an overview of the importance of the sarcolemmal dystrophin-glycoprotein complex and the wider dystrophin complexome in skeletal muscle and its linkage to depolarisation-induced calcium-release mechanisms and the excitation-contraction-relaxation cycle. Besides chronic inflammation, fat substitution and reactive myofibrosis, a major pathobiochemical hallmark of X-linked muscular dystrophy is represented by the chronic influx of calcium ions through the damaged plasmalemma in conjunction with abnormal intracellular calcium fluxes and buffering. Impaired calcium handling proteins should therefore be included in an improved biomarker signature of Duchenne muscular dystrophy.

Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription

Duchenne muscular dystrophy is a severe neuromuscular disease causing a progressive muscle wasting due to mutations in the DMD gene that lead to the absence of dystrophin protein. Adeno-associated virus (AAV)-based therapies aiming to restore dystrophin in muscles, by either exon skipping or microdystrophin expression, are very promising. However, the absence of dystrophin induces cellular perturbations that hinder AAV therapy efficiency. We focused here on the impact of the necrosis-regeneration process leading to nuclear centralization in myofiber, a common feature of human myopathies, on AAV transduction efficiency. We generated centronucleated myofibers by cardiotoxin injection in wild-type muscles prior to AAV injection. Intramuscular injections of AAV1 vectors show that transgene expression was drastically reduced in regenerated muscles, even when the AAV injection occurred 10 months post-regeneration. We show also that AAV genomes were not lost from cardiotoxin regenerated muscle and were properly localised in the myofiber nuclei but were less transcribed leading to muscle transduction defect. A similar defect was observed in muscles of the DMD mouse model mdx. Therefore, the regeneration process per se could participate to the AAV-mediated transduction defect observed in dystrophic muscles which may limit AAV-based therapies.

Revertant Phenomenon in DMD and LGMD2I and Its Therapeutic Implications: A Review of Study Under Mentorship of Terrence Partridge.

This review recollects my initial research focus on revertant fibers (expressing dystrophin in the background of frame-shifting mutation) in Duchenne muscular dystrophy (DMD) muscles in Professor Terrence Partridge’s Muscle Cell Biology Laboratory in MRC Clinical Research Science Center, Harmmersmith Hospital, London, UK. Our data indicated that revertant fibers are most likely resulted from epigenetic random events which skip exon(s) flanking the mutated exon, leading to the restoration of the reading frame. Some of these events establish themselves as relatively permanent skipping patterns, a mechanism similar to multiple transcript species established in various cell types. With this hypothesis, antisense oligonucleotide-mediated exon skipping is likely to have a great chance to achieve restoration of therapeutic levels of dystrophin in DMD muscles. This leads to our first reports of local and systemic efficacy of antisense oligonucleotide-mediated exon skipping for DMD treatment. The experience under Terry’s mentorship shaped my thinking and led me to explore another revertant feature in the dystroglycanopathy caused by mutations in the Fukutin Related Protein (FKRP) gene which functions as a glycosyltransferase. Mutant FKRPs retain partial function and produce a fraction of normal to no detectable levels of laminin-binding α-dystroglycan (matriglycan) in most of the muscle fibers. Reversion to near normal levels of matriglycan expression in muscles with FKRP mutations depends on muscle regeneration and in muscles of neonate mice, suggesting that changes in metabolism and gene expression could be sufficient to compensate for the reduced function of mutant FKRP genes even those associated with severe congenital muscular dystrophy (CMD). This is now supported by our successful demonstration that supply of FKRP mutant mice with ribitol, a precursor for substrate of FKRP, is sufficient to restore the levels of matriglycan with therapeutic significance. Our data overall suggest that rare events of reversion in muscular dystrophy, and likely other diseases could provide unique insight for mechanisms and therapeutic exploitation.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Genetic correction strategies for Duchenne muscular dystrophy and their impact on the heart

BackgroundDuchenne muscular dystrophy (DMD) is an X-linked recessive disorder with early childhood onset characterized by profound loss of muscle strength and associated cardiomyopathy. DMD affects is most often caused by deletions involving single or multiple exons that disrupt the open reading frame of the DMD gene. Mutations causing loss or premature truncation of dystrophin result in dystrophin protein deficiency, which renders the plasma membrane of skeletal myofibers and cardiomyocytes weakened. Aim of reviewGenetic correction is in use to treat DMD since several drugs have been already approved which partially restore dystrophin production through the use of antisense oligonucleotides. There are multiple ongoing clinical trials to evaluate the efficacy of treating DMD with micro-dystrophins delivered by adeno-associated viruses. Future approaches entail gene editing to target the single copy of the DMD gene on the X-chromosome. The primary, near-term goal is restoration of skeletal muscle dystrophin, and for some of these treatments, the efficacy in the heart is not fully known. Here, we discuss the anticipated cardiac outcomes of dystrophin-targeted therapies, and how this information informs genomic medicine for cardiomyopathies, especially in childhood. Key scientific concepts of reviewMany genetic treatment strategies are being implemented to treat DMD. Since most preclinical testing has focused on skeletal muscle, there is a gap in knowledge about the expected effects of these approaches on cardiac genetic correction and cardiomyopathy progression in DMD. Additional study is needed.