Investigating the Potential for Sulforaphane to Attenuate Gastrointestinal Dysfunction in mdx Dystrophic Mice

Gastrointestinal (GI) dysfunction is an important, yet understudied condition associated with Duchenne muscular dystrophy (DMD), with patients reporting bloating, diarrhea, and general discomfort, contributing to a reduced quality of life. In the mdx mouse, the most commonly used mouse model of DMD, studies have confirmed GI dysfunction (reported as altered contractility and GI transit through the small and large intestine), associated with increased local and systemic inflammation. Sulforaphane (SFN) is a natural isothiocyanate with anti-inflammatory and anti-oxidative properties via its activation of Nrf2 signalling that has been shown to improve aspects of the skeletal muscle pathology in dystrophic mice. Whether SFN can similarly improve GI function in muscular dystrophy was unknown. Video imaging and spatiotemporal mapping to assess gastrointestinal contractions in isolated colon preparations from mdx and C57BL/10 mice revealed that SFN reduced contraction frequency when administered ex vivo, demonstrating its therapeutic potential to improve GI function in DMD. To confirm this in vivo, four-week-old male C57BL/10 and mdx mice received vehicle (2% DMSO/corn oil) or SFN (2 mg/kg in 2% DMSO/corn oil) via daily oral gavage five days/week for 4 weeks. SFN administration reduced fibrosis in the diaphragm of mdx mice but did not affect other pathological markers. Gene and protein analysis revealed no change in Nrf2 protein expression or activation of Nrf2 signalling after SFN administration and oral SFN supplementation did not improve GI function in mdx mice. Although ex vivo studies demonstrate SFN’s therapeutic potential for reducing colon contractions, in vivo studies should investigate higher doses and/or alternate routes of administration to confirm SFN’s potential to improve GI function in DMD.

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Electrical impedance myography for the in vivo and ex vivo assessment of muscular dystrophy (mdx ) mouse muscle

Muscle & Nerve ◽

10.1002/mus.24086 ◽

2014 ◽

Vol 49 (6) ◽

pp. 829-835 ◽

Cited By ~ 29

Author(s):

Jia Li ◽

Tom R. Geisbush ◽

Glenn D. Rosen ◽

Jennifer Lachey ◽

Aaron Mulivor ◽

...

Keyword(s):

Muscular Dystrophy ◽

Electrical Impedance ◽

Ex Vivo ◽

Mdx Mouse ◽

Mouse Muscle

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Soluble guanylate cyclase stimulation mitigates skeletal and cardiac muscle dysfunction in a mdx model of Duchenne muscular dystrophy

10.1101/2021.02.14.431156 ◽

2021 ◽

Author(s):

Ling Zhang ◽

Yuanyuan Xu ◽

Keyvan Yousefi ◽

Camila I. Irion ◽

Roger A. Alvarez ◽

...

Keyword(s):

Nitric Oxide ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Signal Propagation ◽

Guanosine Monophosphate ◽

Muscle Pathology ◽

Mdx Mice

AbstractThe impairment of neuronal nitric oxide synthase (nNOS) signaling contributes to disease pathology in the muscle wasting disorder Duchenne muscular dystrophy (DMD). nNOS signal propagation occurs through nitric oxide sensitive soluble guanylate cyclase (sGC), a critical source of cyclic guanosine monophosphate (cGMP) in muscle. Although both nNOS and sGC activity are impaired in DMD patients, little is known about sGC as a therapeutic target. In this study, we tested the hypothesis that stimulating sGC activity with the allosteric agonist BAY41-8543 mitigates striated muscle pathology in the mdx4cv mouse model of DMD. In contrast to DMD patients, mdx mice exhibited greater basal sGC activity than wild type controls with preservation of cGMP levels due partly to upregulation of sGC in some muscles. Stimulating sGC activity in mdx mice with BAY41-8543 substantially reduced skeletal muscle damage, macrophage densities and inflammation and significantly increased resistance to contraction-induced fatigue. BAY41-8543 also enhanced in vivo diaphragm function while reducing breathing irregularities suggesting improved respiratory function. BAY41-8543 attenuated cardiac hypertrophic remodeling, fibrosis and diastolic dysfunction including left atrium enlargement in aged mdx mice. Overall, sGC stimulation significantly mitigated skeletal and cardio-respiratory dysfunction in mdx4cv mice. Importantly, this study provides compelling pre-clinical evidence supporting sGC as a novel target in DMD and the repurposing of FDA-approved sGC stimulators, such as riociguat and veraciguat, as a novel therapeutic approach for DMD.

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Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy

PLoS ONE ◽

10.1371/journal.pone.0245397 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245397

Author(s):

Antonietta Mele ◽

Paola Mantuano ◽

Adriano Fonzino ◽

Francesco Rana ◽

Roberta Francesca Capogrosso ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Ex Vivo ◽

Eccentric Contraction ◽

Invasive Technique ◽

Mdx Mouse ◽

Early Stages ◽

Over Time

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients’ condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages.

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Lmo7 is dispensable for skeletal muscle and cardiac function

AJP Cell Physiology ◽

10.1152/ajpcell.00177.2015 ◽

2015 ◽

Vol 309 (7) ◽

pp. C470-C479 ◽

Cited By ~ 6

Author(s):

Dieu Hung Lao ◽

Mary C. Esparza ◽

Shannon N. Bremner ◽

Indroneal Banerjee ◽

Jianlin Zhang ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Cardiac Function ◽

Cardiac Muscle ◽

Mapk Pathway ◽

Mdx Mouse ◽

Mdx Mice ◽

Mouse Line

Emery-Dreifuss muscular dystrophy (EDMD) is a degenerative disease primarily affecting skeletal muscles in early childhood as well as cardiac muscle at later stages. EDMD is caused by a number of mutations in genes encoding proteins associated with the nuclear envelope (e.g., Emerin, Lamin A/C, and Nesprin). Recently, a novel protein, Lim-domain only 7 ( lmo7) has been reported to play a role in the molecular pathogenesis of EDMD. Prior in vitro and in vivo studies suggested the intriguing possibility that Lmo7 plays a role in skeletal or cardiac muscle pathophysiology. To further understand the in vivo role of Lmo7 in striated muscles, we generated a novel Lmo7-null ( lmo7−/−) mouse line. Using this mouse line, we examined skeletal and cardiac muscle physiology, as well as the role of Lmo7 in a model of muscular dystrophy and regeneration using the dystrophin-deficient mdx mouse model. Our results demonstrated that lmo7−/− mice had no abnormalities in skeletal muscle morphology, physiological function, or regeneration. Cardiac function was also unaffected. Moreover, we found that ablation of lmo7 in mdx mice had no effect on the observed myopathy and muscular regeneration exhibited by mdx mice. Molecular analyses also showed no changes in dystrophin complex factors, MAPK pathway components, and Emerin levels in lmo7 knockout mice. Taken together, we conclude that Lmo7 is dispensable for skeletal muscle and cardiac physiology and pathophysiology.

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β-Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation

Biomolecules ◽

10.3390/biom11111742 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1742

Author(s):

Paola Mantuano ◽

Brigida Boccanegra ◽

Elena Conte ◽

Michela De Bellis ◽

Santa Cirmi ◽

...

Keyword(s):

Mechanism Of Action ◽

Ex Vivo ◽

Mdx Mouse ◽

Mdx Mice ◽

Partial Recovery ◽

Glycoprotein Complex ◽

Promising Therapeutic Target ◽

Potential Synergy ◽

Dystrophin Glycoprotein Complex

ROS-activated cSrc tyrosine kinase (TK) promotes the degradation of β-dystroglycan (β-DG), a dystrophin-glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc-TK represents a promising therapeutic target. In mdx mice, a 4-week subcutaneous treatment with dasatinib (DAS), a pan-Src-TKs inhibitor approved as anti-leukemic agent, increased muscle β-DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)-β-cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of β-DG in pathology progression, meanwhile confirming DAS mechanism of action over the long-term, along with its efficacy and tolerability. The 4-week old mdx mice underwent a 12-week treatment with DAS/HP-β-CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease-relevant readouts. Oral DAS/HP-β-CD efficiently distributed in mdx mice plasma and tissues in a dose-related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing β-DG phosphorylation and restoring its levels dose-dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric-suitable DAS formulation for proper exposure and safety and for enhancing β-DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In-depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies.

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Therapeutic potential of microRNA in tendon injuries

British Medical Bulletin ◽

10.1093/bmb/ldaa002 ◽

2020 ◽

Vol 133 (1) ◽

pp. 79-94 ◽

Cited By ~ 5

Author(s):

Lorenzo Giordano ◽

Giovanna Della Porta ◽

Giuseppe M Peretti ◽

Nicola Maffulli

Keyword(s):

Extracellular Matrix ◽

Therapeutic Potential ◽

Ex Vivo ◽

Tendon Healing ◽

Tendon Injuries ◽

In Vivo Studies ◽

Proliferation And Differentiation ◽

Effective Delivery ◽

Clinical Potential

Abstract Introduction The regulatory role of microRNA (miRNA) in several conditions has been studied, but their function in tendon healing remains elusive. This review summarizes how miRNAs are related to the pathogenesis of tendon injuries and highlights their clinical potential, focusing on the issues related to their delivery for clinical purposes. Sources of data We searched multiple databases to perform a systematic review on miRNA in relation to tendon injuries. We included in the present work a total of 15 articles. Areas of agreement The mechanism of repair of tendon injuries is probably mediated by resident tenocytes. These maintain a fine equilibrium between anabolic and catabolic events of the extracellular matrix. Specific miRNAs regulate cytokine expression and orchestrate proliferation and differentiation of stromal cell lines involved in the composition of the extracellular matrix. Areas of controversy The lack of effective delivery systems poses serious obstacles to the clinical translation of these basic science findings. Growing point In vivo studies should be planned to better explore the relationship between miRNA and tendon injuries and evaluate the most suitable delivery system for these molecules. Areas timely for developing research Investigations ex vivo suggest therapeutic opportunities of miRNA for the management of tendon injuries. Given the poor pharmacokinetic properties of miRNAs, these must be delivered by an adequate adjuvant transport system.

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Contractile efficiency of dystrophic mdx mouse muscle: in vivo and ex vivo assessment of adaptation to exercise of functional end points

Journal of Applied Physiology ◽

10.1152/japplphysiol.00776.2015 ◽

2017 ◽

Vol 122 (4) ◽

pp. 828-843 ◽

Cited By ~ 18

Author(s):

Roberta Francesca Capogrosso ◽

Paola Mantuano ◽

Anna Cozzoli ◽

Francesca Sanarica ◽

Ada Maria Massari ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Damage ◽

Functional Adaptation ◽

Mdx Mouse ◽

Mdx Mice ◽

Chronic Exercise ◽

Exercise Protocol ◽

Dystrophic Muscle

Progressive weakness is a typical feature of Duchenne muscular dystrophy (DMD) patients and is exacerbated in the benign mdx mouse model by in vivo treadmill exercise. We hypothesized a different threshold for functional adaptation of mdx muscles in response to the duration of the exercise protocol. In vivo weakness was confirmed by grip strength after 4, 8, and 12 wk of exercise in mdx mice. Torque measurements revealed that exercise-related weakness in mdx mice correlated with the duration of the protocol, while wild-type (WT) mice were stronger. Twitch and tetanic forces of isolated diaphragm and extensor digitorum longus (EDL) muscles were lower in mdx compared with WT mice. In mdx, both muscle types exhibited greater weakness after a single exercise bout, but only in EDL after a long exercise protocol. As opposite to WT muscles, mdx EDL ones did not show any exercise-induced adaptations against eccentric contraction force drop. qRT-PCR analysis confirmed the maladaptation of genes involved in metabolic and structural remodeling, while damage-related genes remained significantly upregulated and angiogenesis impaired. Phosphorylated AMP kinase level increased only in exercised WT muscle. The severe histopathology and the high levels of muscular TGF-β1 and of plasma matrix metalloproteinase-9 confirmed the persistence of muscle damage in mdx mice. Therefore, dystrophic muscles showed a partial degree of functional adaptation to chronic exercise, although not sufficient to overcome weakness nor signs of damage. The improved understanding of the complex mechanisms underlying maladaptation of dystrophic muscle paves the way to a better managment of DMD patients. NEW & NOTEWORTHY We focused on the adaptation/maladaptation of dystrophic mdx mouse muscles to a standard protocol of exercise to provide guidance in the development of more effective drug and physical therapies in Duchenne muscular dystrophy. The mdx muscles showed a modest functional adaptation to chronic exercise, but it was not sufficient to overcome the progressive in vivo weakness, nor to counter signs of muscle damage. Therefore, a complex involvement of multiple systems underlies the maladaptive response of dystrophic muscle.

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The Effect of Vitamin D Supplementation on Skeletal Muscle in the mdx Mouse Model of Duchenne Muscular Dystrophy

Sports ◽

10.3390/sports7050096 ◽

2019 ◽

Vol 7 (5) ◽

pp. 96

Author(s):

Danielle A. Debruin ◽

Nicola Andreacchio ◽

Erik D. Hanson ◽

Cara A. Timpani ◽

Emma Rybalka ◽

...

Keyword(s):

Skeletal Muscle ◽

Vitamin D ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Weakness ◽

Ex Vivo ◽

Fatigue Properties ◽

Muscle Pathology ◽

Mdx Mouse ◽

Force Frequency

Vitamin D (VitD) has shown to be beneficial in reversing muscle weakness and atrophy associated with VitD deficiency. Duchenne muscular dystrophy is characterized by worsening muscle weakness and muscle atrophy, with VitD deficiency commonly observed. This study aimed to investigate the effect of VitD supplementation on dystrophic skeletal muscle. Eight-week old female control (C57BL/10; n = 29) and dystrophic (C57BL/mdx; n = 23) mice were randomly supplemented with one of three VitD enriched diets (1000, 8000 & 20,000 IU/kg chow). Following a four-week feeding period, the extensor digitorum longus (EDL) and soleus muscles contractile and fatigue properties were tested ex vivo, followed by histological analysis. As expected, mdx muscles displayed higher mass yet lower specific forces and a rightward shift in their force frequency relationship consistent with dystrophic pathology. There was a trend for mdx muscle mass to be larger following the 20,000 IU/kg diet, but this did not result in improved force production. Fiber area in the EDL was larger in mdx compared to controls, and there were higher amounts of damage in both muscles, with VitD supplementation having no effect. Four weeks of VitD supplementation did not appear to have any impact upon dystrophic skeletal muscle pathology at this age.

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