Short-term treatment with Uncaria tomentosa aggravates the injury phenotype in mdx mice

INTRODUCTION: Uncaria tomentosa (Willd. ex Roem. & Schult.) DC. (Rubiaceae) or UT is a medicinal plant with antiviral, antimutagenic, anti-inflammatory and antioxidant properties. Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations in the dystrophin gene; this deficiency leads to sarcolemma instability, inflammation, muscle degeneration and fibrosis. OBJECTIVE: Considering the importance of inflammation to dystrophy progression and the anti-inflammatory activity of UT, in the present study we evaluated whether oral administration of UT extract would ameliorate dystrophy in the mdx mice, a DMD model. METHODS: Eight-week-old male mdx mice were submitted to 200 mg/kg body weight daily UT oral administration for 6 weeks. General histopathology was analysed, and muscle tumor necrosis factor α, transforming growth factor-ß, myostatin and osteopontin transcript levels were assessed. The ability of mice to sustain limb tension to oppose their gravitational force was measured. Data were analysed with the unpaired Student's t-test. RESULTS: Morphologically, both untreated and UT-treated animals exhibited internalised nuclei, increased endomysial connective tissue and variations in muscle fibre diameters. Body weight and muscle strength were significantly reduced in the UT-treated animals. Blood creatine kinase was higher in UT-treated compared to untreated animals. In tibialis anterior, myostatin, transcript was more highly expressed in the UT-treated while in the diaphragm muscle, transforming growth factor-ß transcripts were less expressed in the UT-treated. CONCLUSION: While previous studies identified anti-inflammatory, antiproliferative and anticarcinogenic UT effects, the extract indicates worsening of dystrophic muscles phenotype after short-term treatment in mdx mice.

Low-Level Photobiomodulation Therapy Modulates H2O2 Production, TRPC-6, and PGC-1α Levels in the Dystrophic Muscle.

Objective: This study evaluated photobiomodulation therapy (PBMT) effects on the factors involved in mitochondrial biogenesis, on the mitochondrial respiratory complexes, and on the transient receptor potential canonical channels (such as TRPC-1 and TRPC-6) in in vitro (mdx muscle cells) and in vivo studies (gastrocnemius muscle) from mdx mice, the dystrophin-deficient model of Duchenne muscular dystrophy (DMD). Background: There is no successful treatment for DMD, therefore demanding search for new therapies that can improve the muscle role, the quality of life, and the survival of dystrophic patients. Methods: The dystrophic primary muscle cells received PBMT at 0.6 J and 5 J, and the dystrophic gastrocnemius muscle received PBMT at 0.6 J. Results: The dystrophic muscle cells treated with PBMT (0.6 J and 5 J) showed no cytotoxicity and significantly lower levels in hydrogen peroxide (H2O2) production. We also demonstrated, for the first time, the capacity of PBMT, at a low dose (0.6 J), in reducing the TRPC-6 content and in raising the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) content in the dystrophic gastrocnemius muscle. Conclusions: PBMT modulates H2O2 production, TRPC-6, and PGC-1α content in the dystrophic muscle. These results suggest that laser therapy could act as an auxiliary therapy in the treatment of dystrophic patients.

Astaxanthin Ameliorates Worsened Muscle Dysfunction of MDX Mice Fed with a High-Fat Diet through Reducing Lipotoxicity and Regulating Gut Microbiota.

Duchenne muscular dystrophy (DMD), a severe X-linked inherited neuromuscular disease, has a high prevalence of obesity. Obesity exacerbates muscle damage and results in adverse clinical outcomes. Preventing obesity helps DMD patients delay disease progression and improve quality of life. Astaxanthin (AX) is a kind of carotenoid which has antioxidant and anti-adipogenesis effects. In this study, male C57BL/10ScSnDmdmdx/J mice were fed with a normal diet, a high-fat diet (HFD), and an HFD containing AX for 16 weeks, respectively. The results showed that AX significantly increased gastrocnemius fiber cross-section area and grip strength, improved treadmill endurance test and mitochondrial morphology, and reduced muscle triglyceride and malonaldehyde levels compared to the HFD. Lipidomic analysis revealed that AX decreased high levels of triglyceride, diglyceride, ceramides, and wax ester induced by HFD. Gut microbiota analysis indicated that AX supplementation failed to alleviate abnormal microbiota diversity, but increased the relative abundances of Akkermansia, Bifidobacterium, Butyricicoccus, and Staphylococcus. In conclusion, AX was expected to alleviate disease progression associated with obesity in DMD patients by reducing lipotoxicity and increasing the abundance of beneficial bacteria.

Acute toxicity and regenerative dose finding of an extract of Miconia ferruginata DC. in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe disease with no cure caused by a genetic abnormality, promoting progressive muscle degeneration. Corticosteroids are used drugs in treatment associated with adverse effects. The extract of Miconia ferruginata (Melastomataceae) (MF) has demonstrated potent antioxidant and anti-inflammatory potential in vitro. This study used a DMD model (mdx) to determine the toxic dose of this plant and found a possible non-toxic dose with therapeutic effects. The mdx groups received an intraperitoneal injection of 0 (control group), 50, 100, 200, 300, and 2000 mg kg-1 of the aqueous leaf extract following a single-dose acute toxicity protocol and were observed for 14 days. The range of toxicity of the extract and LD50 were determined. Histopathological analysis, the quantification of fibrosis, and immunohistochemical analysis of the tissues were performed. The results demonstrated that 2000 mg kg-1 was highly toxic, inducing histopathological changes in the tissues evaluated, with 100% mortality in 48 hours. The other doses caused no behavioral changes or signs of toxicity. The MF extract led reduction in histopathological changes, fibrosis, and inflammation, a reduction in HSP70 and an increase in MCL-1 proteins. Doses of 50-200 mg kg-1 demonstrated regenerative tissue and anti-inflammatory potential.

The Effect of Uridine on the State of Skeletal Muscles and the Functioning of Mitochondria in Duchenne Dystrophy.

Duchenne muscular dystrophy is caused by the loss of functional dystrophin that secondarily causes systemic metabolic impairment in skeletal muscles and cardiomyocytes. The nutraceutical approach is considered as a possible complementary therapy for this pathology. In this work, we have studied the effect of pyrimidine nucleoside uridine (30 mg/kg/day for 28 days, i.p.), which plays an important role in cellular metabolism, on the development of DMD in the skeletal muscles of dystrophin deficient mdx mice, as well as its effect on the mitochondrial dysfunction that accompanies this pathology. We found that chronic uridine administration reduced fibrosis in the skeletal muscles of mdx mice, but it had no effect on the intensity of degeneration/regeneration cycles and inflammation, pseudohypetrophy, and muscle strength of the animals. Analysis of TEM micrographs showed that uridine also had no effect on the impaired mitochondrial ultrastructure of mdx mouse skeletal muscle. The administration of uridine was found to lead to an increase in the expression of the Drp1 and Parkin genes, which may indicate an increase in the intensity of organelle fission and the normalization of mitophagy. Uridine had little effect on OXPHOS dysfunction in mdx mouse mitochondria, and moreover, it was suppressed in the mitochondria of wild type animals. At the same time, uridine restored the transport of potassium ions and reduced the production of reactive oxygen species; however, this had no effect on the impaired calcium retention capacity of mdx mouse mitochondria. The obtained results demonstrate that the used dose of uridine only partially prevents mitochondrial dysfunction in skeletal muscles during Duchenne dystrophy, though it mitigates the development of destructive processes in skeletal muscles.

Exploring the Therapeutic Potential of Ectoine in Duchenne Muscular Dystrophy: Comparison with Taurine, a Supplement with Known Beneficial Effects in the mdx Mouse.

Duchenne Muscular Dystrophy (DMD) is a debilitating muscle disorder that condemns patients to year-long dependency on glucocorticoids. Chronic glucocorticoid use elicits many unfavourable side-effects without offering satisfying clinical improvement, thus, the search for alternative treatments to alleviate muscle inflammation persists. Taurine, an osmolyte with anti-inflammatory effects, mitigated pathological features in the mdx mouse model for DMD but interfered with murine development. In this study, ectoine is evaluated as an alternative for taurine in vitro in CCL-136 cells and in vivo in the mdx mouse. Pre-treating CCL-136 cells with 0.1 mM taurine and 0.1 mM ectoine prior to exposure with 300 U/mL IFN-γ and 20 ng/mL IL-1ß partially attenuated cell death, whilst 100 mM taurine reduced MHC-I protein levels. In vivo, histopathological features of the tibialis anterior in mdx mice were mitigated by ectoine, but not by taurine. Osmolyte treatment significantly reduced mRNA levels of inflammatory disease biomarkers, respectively, CCL2 and SPP1 in ectoine-treated mdx mice, and CCL2, HSPA1A, TNF-α and IL-1ß in taurine-treated mdx mice. Functional performance was not improved by osmolyte treatment. Furthermore, ectoine-treated mdx mice exhibited reduced body weight. Our results confirmed beneficial effects of taurine in mdx mice and, for the first time, demonstrated similar and differential effects of ectoine.

Cardio-respiratory and phenotypic rescue of dystrophin/utrophin-deficient mice by combination therapy.

Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense oligonucleotides (AOs) are clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body-wide function and phenotypic rescue of dystrophin /utrophin double knock-out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio-respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically-approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.

Effects of moderate aerobic exercise, low-level laser therapy, or their combination on muscles pathology, oxidative stress and irisin levels in the mdx mouse model of Duchenne muscular dystrophy.

This study aimed to investigate how the combined use of low-level laser therapy (LLLT) and exercise, to reduce the possible side effects and/or increase the benefits of exercise, would affect oxidative stress, utrophin, irisin peptide, and skeletal, diaphragmatic, and cardiac muscle pathologies. In our study, 20 mdx mice were divided into four groups. Groups; sedentary and placebo LLLT (SC), sedentary and LLLT (SL), 30-min swimming exercise (Ex), and 30-min swimming exercise and LLLT (ExL). After 8 weeks of swimming exercise, muscle tests, biochemically; oxidative stress index (OSI), utrophin and irisin levels were measured. Skeletal, diaphragmatic and cardiac muscle histopathological scores, skeletal and cardiac muscle myocyte diameters were determined under the light and electron microscope. While only irisin levels were increased in group SL compared to SC, it was determined that OSI, heart muscle histopathological scores decreased and irisin levels increased in both exercise groups (p < 0.05). In addition, in the ExL group, an increase in rotarod and utrophin levels, and a decrease in muscle and diaphragm muscle histopathological scores were observed (p < 0.05). It was determined that the application of swimming exercise in the mdx mouse model increased the irisin level in the skeletal muscle, while reducing the OSI, degeneration in the heart muscle, inflammation and cardiopathy. When LLLT was applied in addition to exercise, muscle strength, skeletal muscle utrophin levels increased, and skeletal and diaphragmatic muscle degeneration and inflammation decreased. In addition, it was determined that only LLLT application increased the level of skeletal muscle irisin.

Phosphorothioate modification improves exon-skipping of antisense oligonucleotides based on sulfonyl phosphoramidates in mdx mouse myotubes.

2'-O-Methyl (2'-OMe) antisense oligonucleotides (AOs) possessing a various number of 4-(trimethylammonio)butylsulfonyl or tosyl phosphoramidates (N+ and Ts-modifications, respectively) instead of a native phosphodiester linkage were designed to skip exon-23 in dystrophin pre-mRNA transcript in mdx mice myotubes. AOs bearing several zwitterionic N+ modifications in the sequence had remarkably increased thermal stability towards complementary mRNA in comparison with 2'-OMe-RNAs having negatively charged Ts and phosphorothioate (PS) linkages. However, only Ts-modified AOs exhibited a similar level of exon skipping in comparison with fully modified PS-containing 2'-OMe-RNA, whereas the exon skipping induced by N+ modified AOs was much lower with no exon-skipping detected for AOs having seven N+ modifications. The level of exon-skipping was improved once Ts and especially N+ moieties were used in combination with PS-modification, most likely through improved cellular and nuclear uptake of AOs. These results provide new insights on expanding the design of novel chemically modified AOs based on phosphate modifications.

Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy.

BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.

Paeonia lactiflora extract improves the muscle function of mdx mice, an animal model of Duchenne muscular dystrophy, via downregulating the high mobility group box 1 protein.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall. is an ethnopharmacological medicine with a long history of human use for treating various inflammatory diseases in many Asian countries. AIM OF THE STUDY: Duchenne muscular dystrophy (DMD) is an X-linked degenerative muscle disease affecting 1 in 3500 males and is characterized by severe muscle inflammation and a progressive decline in muscle function. This study aimed to elucidate the effects of an ethanol extract of the root of Paeonia lactiflora Pall. (PL) on the muscle function in the muscular dystrophy X-linked (mdx) mouse, the most commonly used animal model of DMD. MATERIALS AND METHODS: Male mdx mice and wild-type controls aged 5 weeks were orally treated with PL for 4 weeks. The corticosteroid prednisolone was used as a comparator drug. Muscle strength and motor coordination were assessed via the grip-strength and rotarod tests, respectively. Muscle damage was evaluated via histological examination and assessment of plasma creatine-kinase activity. Proteomic analyses were conducted to identify the muscle proteins whose levels were significantly affected by PL (ProteomeXchange identifier: PXD028886). Muscle and plasma levels of these proteins, and their corresponding mRNAs were measured using western blotting and ELISA, and quantitative reverse transcription-polymerase chain reaction, respectively. RESULTS: The muscle strength and motor coordination of mdx mice were significantly increased by the oral treatment of PL. PL significantly reduced the histological muscle damage and plasma creatine-kinase activity. Proteomic analyses of the muscle showed that PL significantly downregulated the high mobility group box 1 (HMGB1) protein and Toll-like receptor (TLR) 4, thus suppressing the HMGB1-TLR4-NF-κB signaling, in the muscle of mdx mice. Consequently, the muscle levels of proinflammatory cytokines/chemokines, which play crucial roles in inflammation, were downregulated. CONCLUSION: PL improves the muscle function and reduces the muscle damage in mdx mice via suppressing the HMGB1-TLR4-NF-κB signaling and downregulating proinflammatory cytokines/chemokines.

Ursolic acid increases strength in mdx mice model and may decrease fibrosis deposition by tgf-ß downregulation / El ácido ursólico aumenta la resistencia en el modelo de ratones mdx y podría disminuir la deposición de fibrosis mediante la regulación decreciente de tgf-ß

SUMMARY: Dystrophin disfunction results in sarcolemma destabilization, leading muscle cell damage by continuous degeneration cycles and limited regeneration. In muscle dystrophy, caused by dystrophin dysfunction, inflammation, necrosis and fibrosis are pathophysiological muscle function loss characteristics. As a genetic disease, this muscle dystrophy has no cure, however, advances in drug therapy using glucocorticoids can decrease the disease progression. Subsequently, alternative therapies were studied, such as ursolic acid (UA), that inhibits muscle atrophy and increases muscle mass and strength. Herein, we used 10 mg/kg daily supplementation in mdx mice for 4 weeks to evaluate serum creatine phosphokinase (CPK), muscle strength (Kondziela test), muscular organization (histology) and expression of fibrosis related genes (TGF-ß, TNF-α, mstn and ostn). UA supplementation increased muscle morphological organization, motor strength and decreased muscular TGF-ß expression. Altogether, the gene expression profile, histological organization and strength could suggest that UA treatment did not stop the fibrogenesis but decreased its progress.

RESUMEN: La disfunción de la distrofina resulta en la desestabilización del sarcolema, llevando al daño de las células musculares por ciclos continuos de degeneración y regeneración limitada. En la distrofia muscular, debido a la disfunción de la distrofina, la inflamación, la necrosis y la fibrosis, son características fisiopatológicas de la pérdida de la función muscular. Como enfermedad genetica no es possible remediar esta distrofia muscular, sin embargo, los avances en la terapia de medicamentos con glucocorticoides pueden disminuir la progresión de la enfermedad. Se estudiaron terapias alternativas, como el ácido ursólico (UA), que inhibe la atrofia muscular y aumenta la masa y la fuerza muscular. En este estudio, utilizamos una suplementación diaria de 10 mg / kg en ratones mdx durante 4 semanas para evaluar la creatina fosfoquinasa (CPK) sérica, la fuerza muscular (prueba de Kondziela), la organización muscular (histología) y la expresión de genes relacionados con la fibrosis (TGF-ß, TNF- α, mstn y ostn). La suplementación con AU aumentó la organización morfológica muscular, la fuerza motora y la disminución de la expresión muscular de TGF-ß. El perfil de expresión génica, la organización histológica y la fuerza simultáneamente podrían sugerir que el tratamiento con AU no detuvo la fibrogénesis sino que disminuyó su progreso.

Effects of muscle damage on 31 phosphorus magnetic resonance spectroscopy indices of energetic status and sarcolemma integrity in young mdx mice.

31 Phosphorus magnetic resonance spectroscopy (31 P-MRS) has been shown to detect altered energetic status (e.g. the ratio of inorganic phosphate to phosphocreatine: Pi/PCr), intracellular acid-base status, and free intracellular magnesium ([Mg2+ ]) in dystrophic muscle compared with unaffected muscle; however, the causes of these differences are not well understood. The purposes of this study were to examine 31 P-MRS indices of energetic status and sarcolemma integrity in young mdx mice compared with wild-type and to evaluate the effects of downhill running to induce muscle damage on 31 P-MRS indices in dystrophic muscle. In vivo 31 P-MRS spectra were acquired from the posterior hindlimb muscles in young (4-10 weeks of age) mdx (C57BL/10ScSn-DMDmdx) and wild-type (C57BL/10ScSnJ) mice using an 11.1-T MR system. The flux of phosphate from PCr to ATP was estimated by 31 P-MRS saturation transfer experiments. Relative concentrations of high-energy phosphates were measured, and intracellular pH and [Mg2+ ] were calculated. 1 H2 O-T2 was measured using single-voxel 1 H-MRS from the gastrocnemius and soleus using a 4.7-T MR system. Downhill treadmill running was performed in a subset of mice. Young mdx mice were characterized by elevated 1 H2 O-T2 (p < 0.01), Pi/PCr (p = 0.02), PCr to ATP flux (p = 0.04) and histological inflammatory markers (p < 0.05) and reduced (p < 0.01) [Mg2+ ] compared with wild-type. Furthermore, 24 h after downhill running, an increase (p = 0.02) in Pi/PCr was observed in mdx and wild-type mice compared with baseline, and a decrease (p < 0.001) in [Mg2+ ] and a lower (p = 0.048) intracellular [H+ ] in damaged muscle regions of mdx mice were observed, consistent with impaired sarcolemma integrity. Overall, our findings demonstrate that 31 P-MRS markers of energetic status and sarcolemma integrity are altered in young mdx compared with wild-type mice, and these indices are exacerbated following downhill running.

TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD. METHODS: All experiments were conducted in the DMDmdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca2+]c) and SPCa in EDL (Extensor Digitorum Longus) muscle fibers from age-matched WT and DMDmdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis. RESULTS: As expected from the malignant hyperthermia like episodes observed in several DMDmdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca2+]c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMDmdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMDmdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMDmdx. Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase. CONCLUSIONS: All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.

Flavonoids and Omega3 Prevent Muscle and Cardiac Damage in Duchenne Muscular Dystrophy Animal Model.

Nutraceutical products possess various anti-inflammatory, antiarrhythmic, cardiotonic, and antioxidant pharmacological activities that could be useful in preventing oxidative damage, mainly induced by reactive oxygen species. Previously published data showed that a mixture of polyphenols and polyunsaturated fatty acids, mediate an antioxidative response in mdx mice, Duchenne muscular dystrophy animal model. Dystrophic muscles are characterized by low regenerative capacity, fibrosis, fiber necrosis, inflammatory process, altered autophagic flux and inadequate anti-oxidant response. FLAVOmega ß is a mixture of flavonoids and docosahexaenoic acid. In this study, we evaluated the role of these supplements in the amelioration of the pathological phenotype in dystrophic mice through in vitro and in vivo assays. FLAVOmega ß reduced inflammation and fibrosis, dampened reactive oxygen species production, and induced an oxidative metabolic switch of myofibers, with consequent increase of mitochondrial activity, vascularization, and fatigue resistance. Therefore, we propose FLAVOmega ß as food supplement suitable for preventing muscle weakness, delaying inflammatory milieu, and sustaining physical health in patients affected from DMD.

Assessing the Use of the sGC Stimulator BAY-747, as a Potential Treatment for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTRG2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTRG2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTRG2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTRG2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTRG2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.

Complementary NAD+ replacement strategies fail to functionally protect dystrophin-deficient muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). METHODS: Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. RESULTS: Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. CONCLUSIONS: In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.

A genotyping method combining primer competition PCR with HRM analysis to identify point mutations in Duchenne animal models.

Dystrophin-null sapje zebrafish is an excellent model for better understanding the pathological mechanisms underlying Duchenne muscular dystrophy, and it has recently arisen as a powerful tool for high-throughput screening of therapeutic candidates for this disease. While dystrophic phenotype in sapje larvae can be easily detected by birefringence, zebrafish genotyping is necessary for drug screening experiments, where the potential rescue of larvae phenotype is the primary outcome. Genotyping is also desirable during colony husbandry since heterozygous progenitors need to be selected. Currently, sapje zebrafish are genotyped through techniques involving sequencing or multi-step PCR, which are often costly, tedious, or require special equipment. Here we report a simple, precise, cost-effective, and versatile PCR genotyping method based on primer competition. Genotypes can be resolved by standard agarose gel electrophoresis and high-resolution melt assay, the latter being especially useful for genotyping a large number of samples. Our approach has shown high sensitivity, specificity, and reproducibility in detecting the A/T point mutation in sapje zebrafish and the C/T mutation in the mdx mouse model of Duchenne. Hence, this method can be applied to other single nucleotide substitutions and may be further optimized to detect small insertions and deletions. Given its robust performance with crude DNA extracts, our strategy may be particularly well-suited for detecting single nucleotide variants in poor-quality samples such as ancient DNA or DNA from formalin-fixed, paraffin-embedded material.

Investigation of the effect of taurine supplementation on muscle taurine content in the mdx mouse model of Duchenne muscular dystrophy using chemically specific synchrotron imaging.

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle wasting disorder, which currently has no cure. Supplementation with the drug taurine has been shown to offer therapeutic benefit in the mdx model for DMD, however the mechanism by which taurine protects dystrophic muscle is not fully understood. Mdx muscle is deficient in taurine, however it is not known if this deficiency occurs in the extracellular space, in other cells present in the tissue (such as immune cells) or in the myofibre itself. Likewise, the tissue location of taurine enrichment in taurine treated mdx muscle is not known. In this study we applied X-ray absorption near edge spectroscopy (XANES) at the sulfur K-edge in an imaging format to determine taurine distribution in muscle tissue. XANES is the only technique currently capable of imaging taurine directly in muscle tissue, at a spatial resolution approaching myocyte cell size (20-50 µm). Using a multi-modal approach of XANES imaging and histology on the same tissue sections, we show that in mdx muscle, it is the myofibres that are deficient in taurine, and taurine supplementation ameliorates this deficiency. Increasing the taurine content of mdx myofibres was associated with a decrease in myofibre damage (as shown by the percentage of intact myofibres) and inflammation. These data will help drive future studies to better elucidate the molecular mechanisms through which taurine protects dystrophic muscle; they also support the continued investigation of taurine as a therapeutic intervention for DMD.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac ß-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.