Dystrophin-deficient Mdx Mice Research Articles

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or βcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.

A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthesis of dystrophin protein. Antisense oligonucleotides (AOs) targeted to trigger excision of an exon bearing a mutant premature stop codon in the DMD transcript have been shown to skip the mutated exon and partially restore functional dystrophin protein in dystrophin-deficient mdx mice. To fully exploit the therapeutic potential of this method requires highly efficient systemic AO delivery to multiple muscle groups, to modify the disease process and restore muscle function. While systemic delivery of naked AOs in DMD animal models requires high doses and is of relatively poor efficiency, we and others have recently shown that short arginine-rich peptide-AO conjugates can dramatically improve in vivo DMD splice correction. Here we report for the first time that a chimeric fusion peptide (B-MSP-PMO) consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) and conjugated to a morpholino oligomer (PMO) AO directs highly efficient systemic dystrophin splice correction in mdx mice. With very low systemic doses, we demonstrate that B-MSP-PMO restores high-level, uniform dystrophin protein expression in multiple peripheral muscle groups, yielding functional correction and improvement of the mdx dystrophic phenotype. Our data demonstrate proof-of-concept for this chimeric peptide approach in DMD splice correction therapy and is likely to have broad application.

Embryonic myosin is a regeneration marker to monitor utrophin-based therapies for DMD.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. Constitutive utrophin expression, a structural and functional paralogue of dystrophin, can successfully prevent the dystrophic pathology in the dystrophin-deficient mdx mouse model. In dystrophic muscles, utrophin is increased as part of the repair process and localized at the sarcolemma of regenerating myofibers. The presence of developmental myosin such as embryonic myosin (MyHC-emb) and neonatal represents a useful marker of muscle regeneration and a meaningful indicator of muscle damage, which correlates with the clinical severity of milder Becker muscular dystrophy and DMD patients. In the present study, we demonstrate that MyHC-emb is a robust marker of regeneration at different ages and in different skeletal muscles. We also evaluate the correlation between utrophin, dystrophin and MyHC-emb in wild-type (wt) and regenerating dystrophic muscles. Restoration of dystrophin significantly reduced MyHC-emb levels. Similarly, overexpression of utrophin in the transgenic mdx-Fiona mice reduced the number of MyHC-emb positive fibers to wt level, prevented the regenerative process and rescued the muscle function. In contrast, the absence of utrophin in the dystrophin-deficient double-knockout mice resulted in a higher MyHC-emb content and in a more severe dystrophic pathophysiology than in mdx mice. These data illustrate the importance of monitoring utrophin and MyHC-emb levels in the preclinical evaluation of therapies and provide translational support for the use of developmental myosin as a disease biomarker in DMD clinical trials.

The Structural and Functional Characteristics of the Motor End Plates of Dysferlin-Deficient Mice

The molecular mechanisms that underlie neuromuscular junction plasticity are complex and remain to be fully elucidated. Experimental models of various forms of impaired motor activity may be promising for their study. The dysferlin protein plays a key role in the multimolecular complex, which maintains sarcolemma integrity and the functioning of skeletal muscle cells. We studied the structural and functional characteristics of the diaphragm muscle motor end plates of dysferlin-deficient Bla/J mice (a model of dysferlinopathy), dystrophin-deficient mdx mice (a model of Duchenne muscular dystrophy), and control C57Bl/6 mice. Increased end plate fragmentation and a decrease in the area of individual fragments were observed in mdx mice and absent in Bla/J mice, which indicates a difference in these models of myodystrophy from these characteristics. However, end plates of both mice lines were characterized by a decrease in the density of distribution of nicotinic acetylcholine receptors, as well as by membrane depolarization, presumably, due to altered functional interaction between the α2 isoform of Na,K-ATPase and the nicotinic acetylcholine receptors.

NEW THERAPEUTIC APPROACHES AND THEIR READOUT: O.15Humoral and cell mediated immune response to new dystrophin after morpholino-induced exon skipping therapy in dystrophin-deficient mdx mice

NEW THERAPEUTIC APPROACHES AND THEIR READOUT: O.15Humoral and cell mediated immune response to new dystrophin after morpholino-induced exon skipping therapy in dystrophin-deficient mdx mice

DMD TREATMENT: ANIMAL MODELS: P.207Full-length but not truncated osteoprotegerin binds directly to muscle cells and increases rapidly dystrophic muscle force

Clinical studies have shown that osteoporosis and muscle degeneration can occur in tandem following neuromuscular diseases. The receptor-activator of nuclear factor κB (RANK), the receptor-activator of nuclear factor κB ligand (RANKL), and osteoprotegerin (OPG) triad, a key pathway for bone regulation, is involved in Duchenne muscular dystrophy physiopathology. OPG, a decoy receptor for RANKL, the truncated (TR-OPG-Fc) or full-length OPG (FL-OPG-Fc) fused with an Fc fragment of IgG1 can inhibit RANKL/RANK interactions in vivo or in vitro. TR-OPG-Fc contains only 4 cysteine-rich RANKL domains while FL-OPG-Fc contains 4 cysteine-rich RANKL domains, 2 TRAIL death domains and 1 heparin-binding domain. We have shown that a 10 day treatment with FL-OPG-Fc during the first acute phase of degeneration in dystrophin-deficient mdx mice completely restored force and resistance to eccentric contractions of fast-twitch muscle. Because FL-OPG-Fc was superior to muscle specific deletion of RANK or anti-RANKL, anti-TRAIL, TR-OPG-Fc treatments, we hypothesized that the heparin-binding domain found in FL-OPG-Fc interacts directly with muscle. WB and confocal microscopy demonstrated that FL-OPG-Fc, but not TR-OPG-Fc, binds to C2C12 myotube. We next showed that heparinase III, which cleaves heparan sulfate chains, reduces FL-OPG-Fc binding by 50%. To further support the importance of the heparin domain, FL-OPG-Fc was co-incubated with equal concentration of heparin, which abrogated by 85% its binding ability. Next, using ex vivo contractile properties of isolated fast-twitch EDL, we showed that a 24h FL-OPG-Fc treatment increased significantly the absolute and specific forces of dystrophic EDL. In conclusion, FL-OPG-Fc, through its heparin-binding domain, binds directly muscle cells and can rapidly increase absolute and specific maximal tetanic forces. Current investigations are directed towards a better understanding of the mechanisms by which FL-OPG-Fc improves dystrophic muscles.

Exosome-Derived Dystrophin from Allograft Myogenic Progenitors Improves Cardiac Function in Duchenne Muscular Dystrophic Mice.

Progressive cardiomyocyte loss in Duchenne muscular dystrophy (DMD) leads to cardiac fibrosis, cardiomyopathy, and eventually heart failure. In the present study, we observed that myogenic progenitor cells (MPC) carry mRNA for the dystrophin gene. We tested whether cardiac function can be improved in DMD by allograft transplantation of MPC-derived exosomes (MPC-Exo) into the heart to restore dystrophin protein expression. Exo from C2C12 cells (an MPC cell line) or vehicle were delivered locally into the hearts of MDX mice. After 2days of treatment, we observed that MPC-Exo restored dystrophin expression in the hearts of MDX mice, which correlated with improved myocardial function in dystrophin-deficient MDX mouse hearts. In conclusion, this study demonstrated that allogeneic WT-MPC-Exo transplantation transiently restored dystrophin gene expression and improved cardiac function in MDX mice, suggesting that allogenic exosomal delivery may serve as an alternative treatment for cardiomyopathy of DMD.

Exacerbation of dystrophic cardiomyopathy by phospholamban deficiency mediated chronically increased cardiac Ca2+ cycling in vivo.

Cardiomyopathy is a significant contributor to morbidity and mortality in Duchenne muscular dystrophy (DMD). Membrane instability, leading to intracellular Ca2+ mishandling and overload, causes myocyte death and subsequent fibrosis in DMD cardiomyopathy. On a cellular level, cardiac myocytes from mdx mice have dysregulated Ca2+ handling, including increased resting Ca2+ and slow Ca2+ decay, especially evident under stress conditions. Sarco(endo)plasmic reticulum Ca2+ ATPase and its regulatory protein phospholamban (PLN) are potential therapeutic targets for DMD cardiomyopathy owing to their key role in regulating intracellular Ca2+ cycling. We tested the hypothesis that enhanced cardiac Ca2+ cycling would remediate cardiomyopathy caused by dystrophin deficiency. We used a genetic complementation model approach by crossing dystrophin-deficient mdx mice with PLN knockout (PLNKO) mice [termed double-knockout (DKO) mice]. As expected, adult cardiac myocytes isolated from DKO mice exhibited increased contractility and faster relaxation associated with increased Ca2+ transient peak height and faster Ca2+ decay rate compared with control mice. However, compared with wild-type, mdx, and PLNKO mice, DKO mice unexpectedly had reduced in vivo systolic and diastolic function as measured by echocardiography. Furthermore, Evans blue dye uptake was increased in DKO hearts compared with control, mdx, and PLNKO hearts, demonstrating increased membrane damage, which subsequently led to increased fibrosis in the DKO myocardium in vivo. In conclusion, despite enhanced intracellular Ca2+ handling at the myocyte level, DMD cardiomyopathy was exacerbated owing to unregulated chronic increases in Ca2+ cycling in DKO mice in vivo. These findings have potentially important implications for ongoing therapeutic strategies for the dystrophic heart. NEW & NOTEWORTHY This study examined the effects of phospholamban ablation on the pathophysiology of cardiomyopathy in dystrophin-deficient mice. In this setting, contractility and Ca2+ cycling were enhanced in isolated myocytes; however, in vivo heart function was diminished. Additionally, sarcolemmal integrity was compromised and fibrosis was increased. This is the first study, to our knowledge, examining unregulated Ca2+ cycling in the dystrophin-deficient heart. Results from this study have implications for potential therapies targeting Ca2+ handling in dystrophic cardiomyopathy. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/unregulated-ca2-cycling-exacerbates-dmd-cardiomyopathy/ .

MyD88 is required for satellite cell-mediated myofiber regeneration in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, leads to severe muscle wasting and eventual death of the afflicted individuals, primarily due to respiratory failure. Deficit in myofiber regeneration, potentially due to an exhaustion of satellite cells, is one of the major pathological features of DMD. Myeloid differentiation primary response 88 (MyD88) is an adaptor protein that mediates activation of various inflammatory pathways in response to signaling from Toll-like receptors and interleukin-1 receptor. MyD88 also regulates cellular survival, proliferation and differentiation in a cell-autonomous manner. However, the role of MyD88 in satellite stem cell homeostasis and function in dystrophic muscle remains unknown. In this study, we demonstrate that tamoxifen-inducible deletion of MyD88 in satellite cells causes loss of skeletal muscle mass and strength in the mdx mouse model of DMD. Satellite cell-specific deletion of MyD88 inhibits myofiber regeneration and stimulates fibrogenesis in dystrophic muscle of mdx mice. Deletion of MyD88 also reduces the number of satellite cells and inhibits their fusion with injured myofibers in dystrophic muscle of mdx mice. Ablation of MyD88 in satellite cells increases the markers of M2 macrophages without having any significant effect on M1 macrophages and expression of inflammatory cytokines. Finally, we found that satellite cell-specific deletion of MyD88 leads to aberrant activation of Notch and Wnt signaling in skeletal muscle of mdx mice. Collectively, our results demonstrate that MyD88-mediated signaling in satellite cells is essential for the regeneration of injured myofibers in dystrophic muscle of mdx mice.

Mineralocorticoid Receptor Antagonists in Muscular Dystrophy Mice During Aging and Exercise.

Mineralocorticoid receptor antagonists added to angiotensin converting enzyme inhibitors have shown preclinical efficacy for both skeletal and cardiac muscle outcomes in young sedentary dystrophin-deficient mdx mice also haploinsufficient for utrophin, a Duchenne muscular dystrophy (DMD) model. The mdx genotypic DMD model has mild pathology, making non-curative therapeutic effects difficult to distinguish at baseline. Since the cardiac benefit of mineralocorticoid receptor antagonists has been translated to DMD patients, it is important to optimize potential advantages for skeletal muscle by further defining efficacy parameters. We aimed to test whether therapeutic effects of mineralocorticoid receptor antagonists added to angiotensin converting enzyme inhibitors are detectable using three different reported methods of exacerbating the mdx phenotype. We tested treatment with lisinopril and the mineralocorticoid receptor antagonist spironolactone in: 10 week-old exercised, 1 year-old sedentary, and 5 month-old isoproterenol treated mdx mice and performed comprehensive functional and histological measurements. None of the protocols to exacerbate mdx phenotypes resulted in dramatically enhanced pathology and no significant benefit was observed with treatment. Since endogenous mineralocorticoid aldosterone production from immune cells in dystrophic muscle may explain antagonist efficacy, it is likely that these drugs work optimally during the narrow window of peak inflammation in mdx mice. Exercised and aged mdx mice do not display prolific damage and inflammation, likely explaining the absence of continued efficacy of these drugs. Since inflammation is more prevalent in DMD patients, the therapeutic window for mineralocorticoid receptor antagonists in patients may be longer.

Heme Oxygenase-1 Influences Satellite Cells and Progression of Duchenne Muscular Dystrophy in Mice.

Muscle damage in Duchenne muscular dystrophy (DMD) caused by the lack of dystrophin is strongly linked to inflammation. Heme oxygenase-1 (HO-1; Hmox1) is an anti-inflammatory and cytoprotective enzyme affecting myoblast differentiation by inhibiting myomiRs. The role of HO-1 has not been so far well addressed in DMD. In dystrophin-deficient mdx mice, expression of Hmox1 in limb skeletal muscles and diaphragm is higher than in wild-type animals, being consistently elevated from 8 up to 52 weeks, both in myofibers and inflammatory leukocytes. Accordingly, HO-1 expression is induced in muscles of DMD patients. Pharmacological inhibition of HO-1 activity or genetic ablation of Hmox1 aggravates muscle damage and inflammation in mdx mice. Double knockout animals (Hmox1-/-mdx) demonstrate impaired exercise capacity in comparison with mdx mice. Interestingly, in contrast to the effect observed in muscle fibers, in dystrophin-deficient muscle satellite cells (SCs) expression of Hmox1 is decreased, while MyoD, myogenin, and miR-206 are upregulated compared with wild-type counterparts. Mdx SCs demonstrate disturbed and enhanced differentiation, which is further intensified by Hmox1 deficiency. RNA sequencing revealed downregulation of Atf3, MafK, Foxo1, and Klf2 transcription factors, known to activate Hmox1 expression, as well as attenuation of nitric oxide-mediated cGMP-dependent signaling in mdx SCs. Accordingly, treatment with NO-donor induces Hmox1 expression and inhibits differentiation. Finally, differentiation of mdx SCs was normalized by CO, a product of HO-1 activity. Innovation and Conclusions: HO-1 is induced in DMD, and HO-1 inhibition aggravates DMD pathology. Therefore, HO-1 can be considered a therapeutic target to alleviate this disease. Antioxid. Redox Signal. 00, 000-000.

Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy

Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANKmko) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL/RANK interaction. The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity is significantly depressed in dysfunctional and dystrophic muscles and full-length OPG-Fc treatment increased SERCA activity and SERCA-2a expression. These findings demonstrate the superiority of full-length OPG-Fc treatment relative to truncated OPG-Fc, anti-RANKL, anti-TRAIL or muscle RANK deletion in improving dystrophic muscle function, integrity and protection against eccentric contractions. In conclusion, full-length OPG-Fc represents an efficient alternative in the development of new treatments for muscular dystrophy in which a single therapeutic approach may be foreseeable to maintain both bone and skeletal muscle functions.

Fluorescent characterization of amyloid deposits in the kidneys of mdx mice.

An amyloidosis is a group of diseases that occurs when amyloid proteins are deposited in tissues and organs. In this study, we demonstrated, at the first time, the presence of amyloid deposits in the kidneys of dystrophin-deficient mdx mice, which are widely used as an experimental model of Duchenne muscular dystrophy. We examined cases of renal amyloidosis in aged mdx mice using traditional methods for amyloid detection (Congo red and Thioflavin T), as well as a new fluorescent dye, disodium salt of 2,7-(1-amino-4-sulfo- 2-naphthylazo) fluorene (DSNAF). Using these different fluorescent dyes, we confirmed the amyloid structure of protein deposits in kidneys of aged mdx mice. Additionally, we found that fixation method has profound effects on downstream staining procedures, and demonstrated that the application of specific fixative, zinc-ethanolformaldehyde, instead of traditional neutral-buffered formalin allows reducing the background fluorescence. Also we illustrated the usefulness of novel fluorescent dye DSNAF for detection of amyloid deposits in mouse tissues. Taken together, our results indicate the association of amyloid formation with Duchenne muscular dystrophy and provides new opportunities for studying naturally occurring amyloidosis development in tissues also.

Aspects of Respiratory Control in the Dystrophin‐deficient mdx Mouse

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease characterised by skeletal muscle weakness which extends to the respiratory musculature. Although deficits in the diaphragm muscle of dystrophin‐deficient mdx mice are well described, there is a paucity of information pertaining to central control of breathing in dystrophinopathies. We have previously reported hypoventilation in mdx mice with reserved capacity to increase ventilation in response to chemoactivation (Burns et al., 2017). Systemic and muscle inflammation are reported in mdx mice and human DMD. We sought to examine aspects of respiratory system control in the mdx mouse model of DMD including electrical activity of the diaphragm muscle and markers of inflammation in putative phrenic motor nuclei.Young adult (8 week old) wild‐type (WT) and mdx mice were studied. Inspiratory (Ti) and expiratory (Te) durations were assessed in conscious WT and mdx mice using whole‐body plethysmograpghy. Diaphragm EMG activity was assessed in WT and mdx mice using fine concentric needle electrodes in anaesthetised spontaneously breathing mice. Basal EMG and responsiveness to chemoactivation were determined. Diaphragm muscle function was examined ex vivo. Cervical spinal cord (C3–C5 region) segments of WT and mdx mice were examined for immunohistochemical and gene expression analyses.Poincaré analysis of Ti and Te revealed no evidence of perturbed respiratory rhythm in young mdx mice based on short‐ and long‐term variability of breathing. Diaphragm muscle force was severely depressed in mdx diaphragm within the frequency range of both ventilatory and airway protective behaviours. Mdx diaphragm had potentiated EMG activity during chemoactivation compared to WT mice. Dystrophin mRNA is expressed in the C3–C5 region of WT spinal cords and expression is significantly reduced in mdx mice, with no indication of altered inflammatory gene expression. Putative phrenic motornuclei in mdx mice had unaltered expression of microglia and astrocytes compared to WT.Our study revealed a potentiated neural drive to breathe in mdx mice during chemoactivation suggesting compensatory neuroplasticity facilitating respiratory motor output. Although systemic and muscle inflammation are present in mdx mice, there is no evidence of neuroinflammation in the cervical spinal cord (C3–C5) of young mdx mice. If neuroplasticity is a feature of DMD, it may be possible to boost motor facilitation of breathing through safe interventions and thus preserve or limit deficiencies in respiratory capacity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins.

Delivery of miniaturized dystrophin genes via adeno-associated viral vectors is one leading approach in development to treat Duchenne muscular dystrophy. Here we directly compared the functionality of five mini- and micro-dystrophins via skeletal muscle-specific transgenic expression in dystrophin-deficient mdx mice. We evaluated their ability to rescue defects in the microtubule network, passive stiffness and contractility of skeletal muscle. Transgenic mdx mice expressing the short dystrophin isoform Dp116 served as a negative control. All mini- and micro-dystrophins restored elevated detyrosinated α-tubulin and microtubule density of mdx muscle to values not different from C57BL/10, however, only mini-dystrophins restored the transverse component of the microtubule lattice back to C57BL/10. Passive stiffness values in mdx muscles expressing mini- or micro-dystrophins were not different from C57BL/10. While all mini- and micro-dystrophins conferred significant protection from eccentric contraction-induced force loss in vivo and ex vivo compared to mdx, removal of repeats two and three resulted in less protection from force drop caused by eccentric contraction ex vivo. Our data reveal subtle yet significant differences in the relative functionalities for different therapeutic constructs of miniaturized dystrophin in terms of protection from ex vivo eccentric contraction-induced force loss and restoration of an organized microtubule lattice.

Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.

Muscle fibers lacking dystrophin undergo a long-term alteration of Ca2+ homeostasis, partially caused by a leaky Ca2+ release ryanodine (RyR) channel. S48168/ARM210, an RyR calcium release channel stabilizer (a Rycal compound), is expected to enhance the rebinding of calstabin to the RyR channel complex and possibly alleviate the pathologic Ca2+ leakage in dystrophin-deficient skeletal and cardiac muscle. This study systematically investigated the effect of S48168/ARM210 on the phenotype of mdx mice by means of a first proof-of-concept, short (4 wk), phase 1 treatment, followed by a 12-wk treatment (phase 2) performed in parallel by 2 independent laboratories. The mdx mice were treated with S48168/ARM210 at two different concentrations (50 or 10 mg/kg/d) in their drinking water for 4 and 12 wk, respectively. The mice were subjected to treadmill sessions twice per week (12 m/min for 30 min) to unmask the mild disease. This testing was followed by in vivo forelimb and hindlimb grip strength and fatigability measurement, ex vivo extensor digitorum longus (EDL) and diaphragm (DIA) force contraction measurement and histologic and biochemical analysis. The treatments resulted in functional (grip strength, ex vivo force production in DIA and EDL muscles) as well as histologic improvement after 4 and 12 wk, with no adverse effects. Furthermore, levels of cellular biomarkers of calcium homeostasis increased. Therefore, these data suggest that S48168/ARM210 may be a safe therapeutic option, at the dose levels tested, for the treatment of Duchenne muscular dystrophy (DMD).—Capogrosso, R. F., Mantuano, P., Uaesoontrachoon, K., Cozzoli, A., Giustino, A., Dow, T., Srinivassane, S., Filipovic, M., Bell, C., Vandermeulen, J., Massari, A. M., De Bellis, M., Conte, E., Pierno, S., Camerino, G. M., Liantonio, A., Nagaraju, K., De Luca, A. Ryanodine channel complex stabilizer compound S48168/ARM210 as a disease modifier in dystrophin-deficient mdx mice: proof-of-concept study and independent validation of efficacy.

Tempol Supplementation Restores Diaphragm Force and Metabolic Enzyme Activities in mdx Mice.

Duchenne muscular dystrophy (DMD) is characterized by striated muscle weakness, cardiomyopathy, and respiratory failure. Since oxidative stress is recognized as a secondary pathology in DMD, the efficacy of antioxidant intervention, using the superoxide scavenger tempol, was examined on functional and biochemical status of dystrophin-deficient diaphragm muscle. Diaphragm muscle function was assessed, ex vivo, in adult male wild-type and dystrophin-deficient mdx mice, with and without a 14-day antioxidant intervention. The enzymatic activities of muscle citrate synthase, phosphofructokinase, and lactate dehydrogenase were assessed using spectrophotometric assays. Dystrophic diaphragm displayed mechanical dysfunction and altered biochemical status. Chronic tempol supplementation in the drinking water increased diaphragm functional capacity and citrate synthase and lactate dehydrogenase enzymatic activities, restoring all values to wild-type levels. Chronic supplementation with tempol recovers force-generating capacity and metabolic enzyme activity in mdx diaphragm. These findings may have relevance in the search for therapeutic strategies in neuromuscular disease.

Glucocorticoids Improve Myogenic Differentiation In Vitro by Suppressing the Synthesis of Versican, a Transitional Matrix Protein Overexpressed in Dystrophic Skeletal Muscles.

In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfβ1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration.

Utrophin Compensates dystrophin Loss during Mouse Spermatogenesis

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder resulting from mutations in the dystrophin gene. The mdx/utrn−/− mouse, lacking in both dystrophin and its autosomal homologue utrophin, is commonly used to model the clinical symptoms of DMD. Interestingly, these mice are infertile but the mechanisms underlying this phenomenon remain unclear. Using dystrophin deficient mdx mouse and utrophin haplodeficient mdx/utrn+/− mouse models, we demonstrate the contribution of Dp427 (full-length dystrophin) and utrophin to testis and epididymis development, as well as spermatogenesis. We show that Dp427 deficiency disturbed the balance between proliferation and apoptosis of germ cells during spermatogenesis, which was further disrupted with utrophin haplodeficiency, deciphering a compensatory role of utrophin for dystrophin in the male reproductive system. In the spermatozoa, we have found a compensatory response of utrophin to dystrophin deficiency - namely the upregulation and relocation of utrophin to the flagellar midpiece. This study demonstrates the contribution of Dp427 and utrophin in male fertility, suggesting a potential pathology in DMD patients.

Increased constitutive nitric oxide production by whole body periodic acceleration ameliorates alterations in cardiomyocytes associated with utrophin/dystrophin deficiency

Duchenne Muscular Dystrophy (DMD) cardiomyopathy is a progressive lethal disease caused by the lack of the dystrophin protein in the heart. The most widely used animal model of DMD is the dystrophin-deficient mdx mouse; however, these mice exhibit a mild dystrophic phenotype with heart failure only late in life. In contrast, mice deficient for both dystrophin and utrophin (mdx/utrn−/−, or dKO) can be used to model severe DMD cardiomyopathy where pathophysiological indicators of heart failure are detectable by 8–10weeks of age. Nitric oxide (NO) is an important signaling molecule involved in vital functions of regulating rhythm, contractility, and microcirculation of the heart, and constitutive NO production affects the function of proteins involved in excitation-contraction coupling. In this study, we explored the efficacy of enhancing NO production as a therapeutic strategy for treating DMD cardiomyopathy using the dKO mouse model of DMD. Specifically, NO production was induced via whole body periodic acceleration (pGz), a novel non-pharmacologic intervention which enhances NO synthase (NOS) activity through sinusoidal motion of the body in a headward-footward direction, introducing pulsatile shear stress to the vascular endothelium and cardiomyocyte plasma membrane. Male dKO mice were randomized at 8weeks of age to receive daily pGz (480cpm, Gz±3.0m/s2, 1h/d) for 4weeks or no treatment, and a separate age-matched group of WT animals (pGz-treated and untreated) served as non-diseased controls. At the conclusion of the protocol, cardiomyocytes from untreated dKO animals had, respectively, 4.3-fold and 3.5-fold higher diastolic resting concentration of Ca2+ ([Ca2+]d) and Na+ ([Na+]d) compared to WT, while pGz treatment significantly reduced these levels. For dKO cardiomyocytes, pGz treatment also improved the depressed contractile function, decreased oxidative stress, blunted the elevation in calpain activity, and mitigated the abnormal increase in [Ca2+]d upon mechanical stress. These improvements culminated in a significant reduction in circulating cardiac troponin T (cTnT) and an extension of the median lifespan of dKO mice from 16 to 31weeks. Treatment with L-NAME (NOS inhibitor) significantly decreased overall lifespan and abolished the cardioprotective properties elicited by pGz. Our results provide evidence that enhancement of NO synthesis by pGz can ameliorate cellular dysfunction in dKO cardiomyocytes and may represent a novel therapeutic intervention in DMD cardiomyopathy patients.