Duchenne Muscular Dystrophy Mouse Research Articles

MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin.

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow, hampering muscle repair and regeneration. Irrespective of the success of gene restoration, functional improvement is limited without reducing fibrosis. The levels of miR-29c, a known regulator of collagen, are reduced in DMD. Our goal is to develop translational, antifibrotic therapy by overexpressing miR-29c. We injected the gastrocnemius muscle with either self-complementary AAV.CMV.miR-29c or single-stranded AAV.MCK.micro-dystrophin alone or in combination in the mdx/utrn+/- mouse, a DMD mouse model. Treatment of 3-month-old mdx/utrn+/- mice with AAV.miR-29c showed a reduction in collagen and increased absolute and specific force compared with untreated animals, but neither parameter reached WT levels. Combinatorial gene delivery in 3-month-old mdx/utrn+/- mice further decreased fibrosis, and showed a reduction of transcript levels for Col1A, Col3A, fibronectin, and Tgfb1. In addition, absolute and specific force was normalized and equivalent to WT. However, protection against eccentric contraction fell short of WT levels at this time point. When this same mouse model was treated with miR-29c/micro-dystrophin combinatorial therapy at 1 month of age, there was complete normalization of specific and absolute force and protection against eccentric contraction-induced injury was comparable to WT. These findings highlight the potential for miR-29c as an important addition to the armamentarium for translational gene therapy, especially when used in combination with micro-dystrophin in DMD.

Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle.

Mineralocorticoid and glucocorticoid receptors are closely related steroid hormone receptors that regulate gene expression through many of the same hormone response elements. However, their transcriptional activities and effects in skeletal muscles are largely unknown. We recently identified mineralocorticoid receptors (MR) in skeletal muscles after finding that combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and MR antagonist spironolactone was therapeutic in Duchenne muscular dystrophy mouse models. The glucocorticoid receptor (GR) agonist prednisolone is the current standard-of-care treatment for Duchenne muscular dystrophy because it prolongs ambulation, likely due to its anti-inflammatory effects. However, data on whether glucocorticoids have a beneficial or detrimental direct effect on skeletal muscle are controversial. Here, we begin to define the gene expression profiles in normal differentiated human skeletal muscle myotubes treated with MR and GR agonists and antagonists. The MR agonist aldosterone and GR agonist prednisolone had highly overlapping gene expression profiles, supporting the notion that prednisolone acts as both a GR and MR agonist that may have detrimental effects on skeletal muscles. Co-incubations with aldosterone plus either nonspecific or selective MR antagonists, spironolactone or eplerenone, resulted in similar numbers of gene expression changes, suggesting that both drugs can block MR activation to a similar extent. Eplerenone treatment alone decreased a number of important muscle-specific genes. This information may be used to develop biomarkers to monitor clinical efficacy of MR antagonists or GR agonists in muscular dystrophy, develop a temporally coordinated treatment with both drugs, or identify novel therapeutics with more specific downstream targets.

ANG1 treatment reduces muscle pathology and prevents a decline in perfusion in DMD mice.

Vascular endothelial growth factor (VEGF) and other pro-angiogenic growth factors have been investigated to enhance muscle tissue perfusion and repair in Duchenne muscular dystrophy (DMD). Current understanding is limited by a lack of functional data following in vivo delivery of these growth factors. We previously used dynamic contrast-enhanced computed tomography to monitor disease progression in murine models of DMD, but no study to date has utilized this imaging technique to assess vascular therapy in a preclinical model of DMD. In the current study, we locally delivered VEGF and ANG1 alone or in combination to dystrophic hind limb skeletal muscle. Using functional imaging, we found the combination treatment as well as ANG1 alone prevented decline in muscle perfusion whereas VEGF alone had no effect compared to controls. These findings were validated histologically as demonstrated by increased alpha-smooth muscle actin-positive vessels in muscles that received either VEGF+ANG1 or ANG1 alone compared to the sham group. We further show that ANG1 alone slows progression of fibrosis compared to either sham or VEGF treatment. The findings from this study shed new light on the functional effects of vascular therapy and suggest that ANG1 alone may be a candidate therapy in the treatment of DMD.

Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy.

In the past decade, the development of two innovative technologies, namely, induced pluripotent stem cells (iPSCs) and the CRISPR Cas9 system, has enabled researchers to model diseases derived from patient cells and precisely edit DNA sequences of interest, respectively. In particular, Duchenne muscular dystrophy (DMD) has been an exemplary monogenic disease model for combining these technologies to demonstrate that genome editing can correct genetic mutations in DMD patient-derived iPSCs. DMD is an X-linked genetic disorder caused by mutations that disrupt the open reading frame of the dystrophin gene, which plays a critical role in stabilizing muscle cells during contraction and relaxation. The CRISPR Cas9 system has been shown to be capable of targeting the dystrophin gene and rescuing its expression in in vitro patient-derived iPSCs and in vivo DMD mouse models. In this review, we highlight recent advances made using the CRISPR Cas9 system to correct genetic mutations and discuss how emerging CRISPR technologies and iPSCs in a combined platform can play a role in bringing a therapy for DMD closer to the clinic.

Renin-angiotensin-aldosterone system inhibitors improve membrane stability and change gene-expression profiles in dystrophic skeletal muscles.

Angiotensin-converting enzyme inhibitors (ACEi) and mineralocorticoid receptor (MR) antagonists are FDA-approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the ACEi lisinopril and the nonspecific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy (DMD) mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current DMD standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared with untreated mice. Short-term treatments of dystrophic mice with specific and nonspecific MR antagonists combined with lisinopril led to overlapping gene-expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic, and chemokine gene expression that was not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in DMD.

Myeloid cells are capable of synthesizing aldosterone to exacerbate damage in muscular dystrophy.

FDA-approved mineralocorticoid receptor (MR) antagonists are used to treat heart failure. We have recently demonstrated efficacy of MR antagonists for skeletal muscles in addition to heart in Duchenne muscular dystrophy mouse models and that mineralocorticoid receptors are present and functional in skeletal muscles. The goal of this study was to elucidate the underlying mechanisms of MR antagonist efficacy on dystrophic skeletal muscles. We demonstrate for the first time that infiltrating myeloid cells clustered in damaged areas of dystrophic skeletal muscles have the capacity to produce the natural ligand of MR, aldosterone, which in excess is known to exacerbate tissue damage. Aldosterone synthase protein levels are increased in leukocytes isolated from dystrophic muscles compared with controls and local aldosterone levels in dystrophic skeletal muscles are increased, despite normal circulating levels. All genes encoding enzymes in the pathway for aldosterone synthesis are expressed in muscle-derived leukocytes. 11β-HSD2, the enzyme that inactivates glucocorticoids to increase MR selectivity for aldosterone, is also increased in dystrophic muscle tissues. These results, together with the demonstrated preclinical efficacy of antagonists, suggest MR activation is in excess of physiological need and likely contributes to the pathology of muscular dystrophy. This study provides new mechanistic insight into the known contribution of myeloid cells to muscular dystrophy pathology. This first report of myeloid cells having the capacity to produce aldosterone may have implications for a wide variety of acute injuries and chronic diseases with inflammation where MR antagonists may be therapeutic.

Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes

ABSTRACTKir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current IK1, which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential IK1 abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that IK1 was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.

Abstract 79: Sarcospan Has a Protective Effect During Development of Cardiac Disease

Sarcospan (SSPN) has an important role in stabilizing sarcolemmal dystrophin- and utrophin-glycoprotein adhesion complexes at the cell membrane. Loss of cell adhesion leads to contraction-induced muscle damage, causing muscle dysfunction and cell death. Recently we have shown a specific role for SSPN in modulating cardiac cell adhesion and physiological function. After transthoracic aortic constriction (TAC), SSPN-null mice transitioned toward failure faster than wild-type (WT) mice. Muscle histology revealed large focal areas of collagen deposition in SSPN-null hearts after TAC compared to WT hearts, suggesting that increased membrane fragility affected cardiomyocyte survival. Our laboratory has shown that SSPN loss reduces sarcolemmal dystrophin levels and associated adhesion complexes in the heart. Whereas, the complete loss of dystrophin leads to development of Duchenne muscular dystrophy (DMD), causing cardiac dysfunction and early mortality. Overexpression of SSPN in DMD mice increased cell adhesion and laminin binding in hearts, leading to improvements in tissue histopathology and increased expression of utrophin, a functional homologue of dystrophin. Examining the restorative potential of SSPN in dystrophic cardiac tissue, led us to query whether compensatory upregulation of SSPN occurs in failing hearts of TAC-treated WT mice. In failing non-DMD hearts, we found that SSPN expression is increased. We have evidence of a chaperone role for SSPN, and its increased expression in the failing heart may contribute to the increased localization of dystrophin and associated glycoprotein complexes at the sarcolemma, which we observed in failing WT hearts compared to untreated controls. The upregulation of cell-stabilizing cell adhesion complexes may compensate for increased wall stress and counter pathological processes that culminate in cardiomyocyte demise, and we are exploring whether naturally increased expression or transgenic overexpression of SSPN in the heart may protect against damage. In summary, we have found that SSPN promotes cardiac function by maintaining cell adhesion and promoting cell survival during disease conditions.

Abstract 255: Role of Connexin 43 in Muscular Dystrophy Cardiomyopathy

Duchenne muscular dystrophy (DMD) is caused by an X-linked mutation that leads to the absence of dystrophin, resulting in life-threatening arrhythmogenesis and associated heart failure. The downstream events that lead to cardiomyopathy are largely unexplored. We unveiled a novel role for the gap junction protein connexin43 (Cx43) in maintaining cardiac conduction and ultimately in preventing heart failure from occurring in DMD. In mild mdx and severe mdx:utr mouse models of DMD, and human DMD tissues, Cx43 was found to be aberrantly upregulated and mislocalized to lateral sides of cardiomyocytes, with an abnormal phosphorylation pattern. We found that a triplet of serine residues, which plays a key role in a variety of cardiac pathologies, was hypophosphorylated in mdx relative to wild-type hearts. Electrocardiography on isoproterenol (Iso) challenged mice showed that both DMD mouse models developed arrhythmias and died within 24 hours, while wild-type mice were free of pathology. Administering peptide mimetic inhibitors of lateralized Cx43 channels prior to Iso challenge protected mdx mice from arrhythmogenesis and death. Cx43 copy number reduction in mdx:Cx43(+/-) mice not only ameliorated acute but also chronic, long-term symptoms of cardiomyopathy in mdx mice. Overall, our findings suggest that Cx43 lateralization contributes significantly to DMD cardiomyopathy and that selective inhibition of misplaced, upregulated channels may provide substantial benefit.

Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models.

Duchenne muscular dystrophy (DMD) is an X-linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin-associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn(+/-) and mdx/utrn(-/-) mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested invivo, with mdx/utrn(-/-) mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation-evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high-rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn(-/-) mice. Overall, we showed mild 'myasthenia-like' neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.

390. Impact of a Treatment with Antioxidant on Gene Transfer Efficiency After Recombinant Adeno-Associated Vector Injection in a Mouse Model of Duchenne Muscular Dystrophy

Recombinant adeno-associated virus (rAAV)-based vectors are promising tools for the treatment of Duchenne muscular dystrophy (DMD) by gene therapy. Following rAAV injection in murine and canine models of DMD, several groups have reported significant phenotype improvements without notable toxicity, raising hope for future clinical translations. However, the long term maintenance of therapeutic benefits is an important, and yet unresolved issue. In previous studies conducted in DMD mice, we and others have demonstrated that rAAV-mediated transgene expression decreases progressively, even when clinically relevant vector doses are injected. Our team described several “restriction” factors having a negative impact on rAAV transduction, notably a loss of vector genomes resulting from muscle cell necrosis but also an oxidative damage affecting transgene mRNA. These first results support the fact that the tissue context in which rAAV vectors are delivered is of critical importance and can significantly affect their efficiency. In addition, they open new avenues for improvement since we can now consider counteracting these restriction phenotypes prior to rAAV injection. In the case of DMD, oxidative stress seems to occupy a central position in both muscle cell pathophysiology and rAAV transgene mRNA degradation. Therefore, we designed an innovative strategy using a relevant antioxidant agent routinely used in human medicine: N-acetylcysteine. DMD mice, pre-treated or not with this compound, were subsequently injected with a rAAV vector carrying a reporter transgene. The transduction efficiency, together with the expression and activity of the transgene, were carefully monitored and compared two months later. The outcome of this innovative approach will certainly pave the way for future combinatorial protocols using pharmacological agents and rAAV vectors in DMD muscles.

624. A Single Neonatal Delivery of an Exon 2 Directed AAV9.U7snRNA Vector Results in Long-Term Dystrophin Expression That Prevents Pathologic Features in the Dup2 Mouse

We recently identified an internal ribosome entry site (IRES) within exon 5 of the DMD gene. Mutations that truncate the reading frame 5’ of this exon can result in use of the IRES for alternate translational initiation beginning within exon 6 that results in expression of an N-truncated isoform. Despite lacking the calponin homology domain 1 (CH1) of the actin binding domain 1 (ABD1), this isoform is highly functional, as demonstrated by the minimal symptoms in patients who express it. Consistent with genotype-phenotype correlations in DMD patients, the IRES is not active in the presence of exon 2 duplication but is active when exon 2 is deleted. We developed an AAV9.U7snRNA vector to that truncates DMD reading frame by skipping of exon 2, and have shown that in a Duchenne muscular dystrophy (DMD) mouse model carrying a duplication of exon 2 (the Dup2 mouse), postnatal intramuscular (IM) or intravascular (IV) treatment results in functional and pathologic improvement in skeletal muscle. Relevant to efforts to identify and treat DMD patients at an earlier age, we sought here to determine whether earlier gene transfer might slow down or even prevent the development of pathology. Dup2 mice were injected via facial vein at postnatal day 1 (P1) with 1E12 total vector genomes of the AAV9.U7snRNA vector and sacrificed at either 1, 3, 6, or 12 months post-injection for evaluation of exon 2 skipping by RT-PCR, quantification of dystrophin expression, and characterization of histopathology. To model the applicability of this approach beyond exon 2 duplication patients, the same vector was used to treat 6 human patient fibroblast-derived transdifferentiated myoblasts (FibroMyoD cells) harboring various mutations within exons 1 to 4. In the Dup2 mouse, efficient skipping and abundant dystrophin expression were present up to one year following the single AAV injection. Dystrophic pathology was absent at all-time points; at one year, less than 1% of fibers showed central nucleation, in comparison to ~70% in untreated Dup2 mice. Two tests on the ex vivo diaphragm preparations: isometric force (providing assessment of strength), and eccentric contractions (evaluating sarcolemma stability) were performed at 3 and 6 months following P1 injection. Both tests demonstrated little to no difference between treated animals and wild type mice. In all FibroMyoD cultures, abundant exon 2 skipping and dystrophin expression were detected in myotubes at 14 days of culture after treatment. These results suggest that this exon-skipping vector offers a therapeutic approach not only to patients with exon 2 duplications but with all mutations within the first four DMD exons (~6% of patients), an area of the gene largely ignored by the current therapeutic approaches. This work strongly supports the idea that early treatment of these patients will have longstanding and significant benefit resulting in a better outcome.

379. MicroRNA-29 and Micro-Dystrophin Combinatorial Therapy Suppresses Fibrosis and Restores Function to mdx/utrn+/− Mice

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in muscle loss and progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow and excludes endomysial derived constituents hampering muscle repair and regeneration. Irrespective of the success of gene restoration (molecular or pharmacologic) functional improvement is limited without reduction of muscle fibrosis. miR-29c regulates collagen levels making it an ideal candidate for decreasing muscle fibrosis. miR-29c levels are reduced in DMD and our goal is to develop an anti-fibrotic therapy by overexpressing miR-29c with adeno-associated virus (AAV) mediated delivery in combination with micro-dystrophin to improve membrane stability. We injected scAAVrh.74.CMV. miR-29c alone, co-delivered with rAAVrh.74.MCK. micro-dystrophin, and rAAVrh.74.MCK. micro-dystrophin alone by intramuscular injection (IM) into the left gastrocnemius (GAS) muscle of 3 month old mdx/utrn+/- mice, a DMD mouse model. GAS muscle was analyzed 3 months post-injection to assess collagen accumulation by Sirius Red staining and subsequent quantification with ImageJ. Additional outcomes included miR-29c and collagen transcript levels, force measurements in the GAS muscle, fiber diameter measurements and western blot analysis for proteins involved in muscle regeneration (MyoD, Myogenin). Analogous to DMD tissue, we demonstrated a significant reduction in miR-29c levels in mdx/utrn+/− muscle correlated with increased fibrosis measured by Sirius red staining. Following 3 months of treatment with scAAV. miR-29c alone, there was a significant reduction in fibrosis (treated-23.5%±1.3 vs. untreated-27.8% ±0.6, p<0.01) in the GAS muscle. When co-delivered with micro-dystrophin we see further reduction in collagen (41%) by Sirius red staining along with significantly reduced mRNA levels of Col1A, Col3A, fibronectin and TGF-β levels. We observed an increase in specific and absolute force in the muscle treated with miR-29c alone compared to the untreated limb, which when combined with micro-dystrophin led to absolute and specific force that were not significantly different than wild-type (miR-29c treated-204.7±11.7 vs. untreated-151.6±14.5 vs. combined-244.2±6.6 vs. wild type-313.1±40.69 p<0.01). We also observed a significant increase in gastroc weight in those muscles that were co-treated. Demonstration of increased fibrosis and decreased miR-29c expression in the mdx/utrn+/− mice and dystrophin-deficient patients validates the mouse model as representative of the human disease. Initial results using AAV. miR-29c as an anti-fibrotic therapy suggest that there is beneficial effect with reduction in collagen levels, a key contributor in fibrosis. Moreover, when combined with micro-dystrophin to improve membrane stability, miR-29 upregulation normalized muscle force. These data provide rationale for overexpression of miR-29c to reduce fibrosis along with dystrophin replacement as a potential treatment for DMD.

380. Therapeutic Capacity of AAV-Delivered Muscle-Specific-Expressed Micro-Utrophin (ΔR4-R21/ΔCT) in mdX4cv Skeletal Muscles

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by dystrophin mutations. Utrophin is a dystrophin paralogue that can prevent necrosis in the mdx mouse DMD model. We designed an expression plasmid containing a miniaturized M-creatine kinase regulatory cassette (CK8e) and a rationally designed micro-utrophinΔR4-21/ΔCT (µUtrn) to accommodate the limited capacity of recombinant adeno-associated virus (rAAV). Both intramuscular (IM) and intravascular (IV) delivery of rAAV6-CK8-µUtrn at 2 weeks of age profoundly mitigated the dystrophic phenotype in skeletal muscles when examined at 3-6 months of age. These improvements were observed in many but not all skeletal muscle histological and functional parameters. At a pathological level µUtrn production was associated with significant reductions in centrally nucleated muscle fibers and with a greatly reduced prevalence of muscle regions containing inflammatory cells. µUtrn production also restored dystrophin-glycoprotein-complex (DGC) components β-Dystroglycan, Δ-Sarcoglycan, α-Dystrobrevin-2, and α1-Syntrophin to the sarcolemma. As expected, nNOS was not restored, presumably because CK8-µUtrn lacks direct nNOS binding sites. Analysis of µUtrn immunostaining intensities 3-months post-treatment suggested lower µUtrn levels in many of the 1, 2a and 2d/x fibers than in the 2b fibers. These differences would be consistent with the relative transcriptional activities of the CK8e regulatory cassette in different adult mouse fiber types: 2b > 2d/x > 2a > 1. Given the important structural role of costameres in connecting the sarcolemma to sarcomeres, we examined utrophin and µUtrn localization patterns and found endogenous utrophin localized in a costameric pattern adjacent to Z-line end points while abutting sarcolemma junctions. However, while µUtrn was also associated with a costameric lattice, its striations were unexpectedly only ~0.8 µm apart compared to ~2.2 µm for endogenous utrophin in untreated mdx4cv muscles. How the presence of two different costameric lattice patterns may affect skeletal muscle function is not known. Since mdx4cv mice are known to exhibit a fragmented synaptic phenotype, we also examined neuromuscular junctions (NMJs) in treated and untreated mice and found that µUtrn prevented synapse fragmentation and also restored the depth of synaptic folds. Interestingly, the NMJs in µUtrn-treated muscles exhibited a more highly branched architecture of the synaptic folds, and this may have compensated for the reduced number of fold openings observed in untreated mice. µUtrn treatment also provided partial restoration of the reduced Achilles myotendinous junction folds seen in mdx4cv mice, and did not lead to either the myotendinous strain injury or the ringed fiber formation associated with micro-dystrophinΔR4-R23/ΔCT mediated therapy. Importantly, physiological studies indicated that IM treated tibialis anterior muscles maintained peak force production and exhibited partially improved specific force production. Overall, rAAV6-CK8-µUtrn treatment of mdx4cv mice provided major improvements in many muscle parameters. However, it did not overcome all dystrophic deficits, and it modified the normal costameric lattice structure. Further improvements might be achieved by expressing µUtrn via regulatory cassettes with higher relative activity in 1, 2a and 2d/x fibers, and by further modifications of µUtrn (ΔR4-R21/ΔCT)'s design.

Duchenne Muscular Dystrophy Mice and Men: Can Understanding a Genetic Cardiomyopathy Inform Treatment of Other Myocardial Diseases?

Duchenne muscular dystrophy mouse models have a predictable and reproducible time course of cardiomyopathy progression with discrete pathogenic steps, which closely parallel what we know occurs in the cardiomyopathy of patients with Duchenne muscular dystrophy. The slow progression of early pathogenic steps common to many cardiomyopathies may make Duchenne muscular dystrophy models useful for identifying novel treatment targets and testing the therapeutic value of new treatment paradigms for an at-risk patient population far beyond those with muscular dystrophies. Dilated cardiomyopathy is a leading cause of death in individuals affected by Duchenne muscular dystrophy (DMD), and nearly all DMD patients develop dilated cardiomyopathy by age 18. DMD and the milder Becker muscular dystrophy result from mutations in the X-linked gene encoding dystrophin, a membrane-associated protein that helps protect striated muscle cell membranes from damage. Clinical signs resulting from lower limb muscle weakness lead to DMD diagnosis typically by 5 years of age and loss of ambulation by 12 years of age. Skeletal myopathy in Becker muscular dystrophy has a later age of onset and slower progression than DMD, and dilated cardiomyopathy is the primary cause of mortality. Earlier ventilatory support for affected respiratory muscles and timely management of pulmonary infections have led to increased survival for DMD patients into their mid-twenties. These advances have made increasingly evident the underlying cardiomyopathy that presents as heart failure and often fatal arrhythmias in the later decades of life. Despite the devastating consequences of DMD cardiomyopathy, its unique features may provide new insights into myocardial disease. First, the consistent lag between evident skeletal myopathy and cardiomyopathy affords early detection and possible treatment of a patient population that is genetically programmed to develop cardiac dysfunction. Second, the myocardial disease exhibits a relatively slow progression, providing the ability to understand each step involved. Third, there are isogenic …

Sarcospan Regulates Cardiac Isoproterenol Response and Prevents Duchenne Muscular Dystrophy-Associated Cardiomyopathy.

BackgroundDuchenne muscular dystrophy is a fatal cardiac and skeletal muscle disease resulting from mutations in the dystrophin gene. We have previously demonstrated that a dystrophin‐associated protein, sarcospan (SSPN), ameliorated Duchenne muscular dystrophy skeletal muscle degeneration by activating compensatory pathways that regulate muscle cell adhesion (laminin‐binding) to the extracellular matrix. Conversely, loss of SSPN destabilized skeletal muscle adhesion, hampered muscle regeneration, and reduced force properties. Given the importance of SSPN to skeletal muscle, we investigated the consequences of SSPN ablation in cardiac muscle and determined whether overexpression of SSPN into mdx mice ameliorates cardiac disease symptoms associated with Duchenne muscular dystrophy cardiomyopathy.Methods and Results SSPN‐null mice exhibited cardiac enlargement, exacerbated cardiomyocyte hypertrophy, and increased fibrosis in response to β‐adrenergic challenge (isoproterenol; 0.8 mg/day per 2 weeks). Biochemical analysis of SSPN‐null cardiac muscle revealed reduced sarcolemma localization of many proteins with a known role in cardiomyopathy pathogenesis: dystrophin, the sarcoglycans (α‐, δ‐, and γ‐subunits), and β1D integrin. Transgenic overexpression of SSPN in Duchenne muscular dystrophy mice (mdx TG) improved cardiomyofiber cell adhesion, sarcolemma integrity, cardiac functional parameters, as well as increased expression of compensatory transmembrane proteins that mediate attachment to the extracellular matrix.Conclusions SSPN regulates sarcolemmal expression of laminin‐binding complexes that are critical to cardiac muscle function and protects against transient and chronic injury, including inherited cardiomyopathy.

TRAF6 regulates satellite stem cell self-renewal and function during regenerative myogenesis.

Satellite cells are a stem cell population within adult muscle and are responsible for myofiber regeneration upon injury. Satellite cell dysfunction has been shown to underlie the loss of skeletal muscle mass in many acquired and genetic muscle disorders. The transcription factor paired box-protein-7 (PAX7) is indispensable for supplementing the reservoir of satellite cells and driving regeneration in normal and diseased muscle. TNF receptor-associated factor 6 (TRAF6) is an adaptor protein and an E3 ubiquitin ligase that mediates the activation of multiple cell signaling pathways in a context-dependent manner. Here, we demonstrated that TRAF6-mediated signaling is critical for homeostasis of satellite cells and their function during regenerative myogenesis. Selective deletion of Traf6 in satellite cells of adult mice led to profound muscle regeneration defects and dramatically reduced levels of PAX7 and late myogenesis markers. TRAF6 was required for the activation of MAPKs ERK1/2 and JNK1/2, which in turn activated the transcription factor c-JUN, which binds the Pax7 promoter and augments Pax7 expression. Moreover, TRAF6/c-JUN signaling repressed the levels of the microRNAs miR-1 and miR-206, which promote differentiation, to maintain PAX7 levels in satellite cells. We also determined that satellite cell-specific deletion of Traf6 exaggerates the dystrophic phenotype in the mdx (a mouse model of Duchenne muscular dystrophy) mouse by blunting the regeneration of injured myofibers. Collectively, our study reveals an essential role for TRAF6 in satellite stem cell function.

P2RX7 purinoceptor: a therapeutic target for ameliorating the symptoms of duchenne muscular dystrophy.

BackgroundDuchenne muscular dystrophy (DMD) is the most common inherited muscle disease, leading to severe disability and death in young men. Death is caused by the progressive degeneration of striated muscles aggravated by sterile inflammation. The pleiotropic effects of the mutant gene also include cognitive and behavioral impairments and low bone density.Current interventions in DMD are palliative only as no treatment improves the long-term outcome. Therefore, approaches with a translational potential should be investigated, and key abnormalities downstream from the absence of the DMD product, dystrophin, appear to be strong therapeutic targets. We and others have demonstrated that DMD mutations alter ATP signaling and have identified P2RX7 purinoceptor up-regulation as being responsible for the death of muscles in the mdx mouse model of DMD and human DMD lymphoblasts. Moreover, the ATP–P2RX7 axis, being a crucial activator of innate immune responses, can contribute to DMD pathology by stimulating chronic inflammation. We investigated whether ablation of P2RX7 attenuates the DMD model mouse phenotype to assess receptor suitability as a therapeutic target.Methods and FindingsUsing a combination of molecular, histological, and biochemical methods and behavioral analyses in vivo we demonstrate, to our knowledge for the first time, that genetic ablation of P2RX7 in the DMD model mouse produces a widespread functional attenuation of both muscle and non-muscle symptoms. In dystrophic muscles at 4 wk there was an evident recovery in key functional and molecular parameters such as improved muscle structure (minimum Feret diameter, p < 0.001), increased muscle strength in vitro (p < 0.001) and in vivo (p = 0.012), and pro-fibrotic molecular signatures. Serum creatine kinase (CK) levels were lower (p = 0.025), and reduced cognitive impairment (p = 0.006) and bone structure alterations (p < 0.001) were also apparent. Reduction of inflammation and fibrosis persisted at 20 mo in leg (p = 0.038), diaphragm (p = 0.042), and heart muscles (p < 0.001). We show that the amelioration of symptoms was proportional to the extent of receptor depletion and that improvements were observed following administration of two P2RX7 antagonists (CK, p = 0.030 and p = 0.050) without any detectable side effects. However, approaches successful in animal models still need to be proved effective in clinical practice.ConclusionsThese results are, to our knowledge, the first to establish that a single treatment can improve muscle function both short and long term and also correct cognitive impairment and bone loss in DMD model mice. The wide-ranging improvements reflect the convergence of P2RX7 ablation on multiple disease mechanisms affecting skeletal and cardiac muscles, inflammatory cells, brain, and bone. Given the impact of P2RX7 blockade in the DMD mouse model, this receptor is an attractive target for translational research: existing drugs with established safety records could potentially be repurposed for treatment of this lethal disease.

Soluble guanylyl cyclase regulates skeletal muscle fiber type plasticity, fatigue resistance and whole body insulin resistance

Pathogenic defects in NO-cGMP signaling drive skeletal muscle dysfunction in Duchenne muscular dystrophy (DMD). These defects arise from decreased nNOS and guanylyl cyclase (GC) activity and loss of spatial control of NO production. Therapeutics such as sildenafil that amplify NO-cGMP signaling reduce skeletal muscle dysfunction in DMD patients and mouse models of DMD making GC and cGMP attractive therapeutic targets. However, GC and cGMP functions in skeletal muscle are poorly defined hindering therapy development. To remove this barrier, we investigated the functions of GC in skeletal muscle. We report that α1β1 soluble GC (sGC) is the primary cGMP source in skeletal muscle. α1β1 sGC expression was greater in oxidative muscles suggesting a greater cGMP synthesis capacity and muscle-specific differences in sGC function. Interestingly, sGC activity exhibited partial nNOS dependence. Analyses of sGC subcellular localization revealed a pool of α1β1 at the cis-Golgi complex in muscle cells. α1β1 and α2β1 sGC localized to the microvasculature. Muscles lacking a functional α1 sGC subunit (α1β1 sGC deficient) exhibited reduced fatigue resistance and normal hypertrophic growth. Surprisingly, α1β1 sGC deficiency had no impact on mitochondrial content suggesting that mitochondrial density may not be as tightly regulated by cGMP as previously thought. α1β1 sGC deficiency had a modest effect on mitochondrial ATP synthesis. Also, loss of α1β1 sGC triggered a type IIA to type IIX fiber shift. Although this shift was unlikely to significantly enhance fatigability, it may impact insulin metabolism because type IIX fiber content positively correlates with insulin insensitivity. Indeed, α1β1 sGC null mice exhibited gender-specific defects in whole body insulin sensitivity consistent with reports of gender-specific effects of cGMP on metabolism but normal glucose tolerance indicating compensatory changes in glucose metabolism. In summary, these findings argue that NO signaling through sGC plays important roles in muscle fatigue and fiber type specification and suggest sGC as a target for mitigating skeletal muscle fatigue in DMD. These data also suggest that disruption of NO-cGMP signaling may contribute to the poorly understood metabolic defects in DMD patients. By showing that reductions in cGMP synthesis promote muscle fatigue, type IIX content and insulin resistance, these data suggest new insights as to how decreases in cGMP may contribute to the development of metabolic dysfunction and disease, particularly over time. Importantly, these results suggest sGC as a potential target for mitigating insulin resistance.

Mineralocorticoid receptors are present in skeletal muscle and represent a potential therapeutic target.

Early treatment with heart failure drugs lisinopril and spironolactone improves skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. The angiotensin converting enzyme inhibitor lisinopril and mineralocorticoid receptor (MR) antagonist spironolactone indirectly and directly target MR. The presence and function of MR in skeletal muscle have not been explored. MR mRNA and protein are present in all tested skeletal muscles from both wild-type mice and DMD mouse models. MR expression is cell autonomous in both undifferentiated myoblasts and differentiated myotubes from mouse and human skeletal muscle cultures. To test for MR function in skeletal muscle, global gene expression analysis was conducted on human myotubes treated with MR agonist (aldosterone; EC50 1.3 nM) or antagonist (spironolactone; IC50 1.6 nM), and 53 gene expression differences were identified. Five differences were conserved in quadriceps muscles from dystrophic mice treated with spironolactone plus lisinopril (IC50 0.1 nM) compared with untreated controls. Genes down-regulated more than 2-fold by MR antagonism included FOS, ANKRD1, and GADD45B, with known roles in skeletal muscle, in addition to NPR3 and SERPINA3, bona fide targets of MR in other tissues. MR is a novel drug target in skeletal muscle and use of clinically safe antagonists may be beneficial for muscle diseases.