Direct observation of failing fibers in muscles of dystrophic mice provides mechanistic insight into muscular dystrophy

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin. Dystrophin's function is not known, but its cellular location and associations with both the force-generating contractile core and membrane-spanning entities suggest a role in mechanically coupling force from its intracellular origins to the fiber membrane and beyond. We report here the presence of destructive contractile activity in lumbrical muscles from dystrophin-deficient ( mdx) mice during nominally quiescent periods following exposure to mechanical stress. The ectopic activity, which was observable microscopically, resulted in longitudinal separation and clotting of fiber myoplasm and was absent when calcium (Ca2+) was removed from the bathing medium. Separation and clotting of myoplasm were also produced in dystrophin-deficient muscles by local application of a Ca2+ ionophore to create membrane breaches in the absence of mechanical stress, whereas muscles from control mice tolerated ionophore-induced entry of Ca2+ without damage. These observations suggest a failure cascade in dystrophin-deficient fibers that 1) is initiated by a stress-induced influx of extracellular Ca2+, causing localized activation to continue after cessation of stimulation, and 2) proceeds as the persistent local activation, combined with reduced lateral mechanical coupling between the contractile core and the extracellular matrix, results in longitudinal separation of myoplasm in nonactivated regions of the fiber. This mechanism invokes both the membrane stabilization and the mechanical coupling functions frequently proposed for dystrophin and suggests that, whereas the absence of either function alone is not sufficient to cause fiber failure, their combined absence is catastrophic.

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Poloxamer 188 reduces the contraction-induced force decline in lumbrical muscles from mdx mice

AJP Cell Physiology ◽

10.1152/ajpcell.00017.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. C146-C150 ◽

Cited By ~ 45

Author(s):

Rainer Ng ◽

Joseph M. Metzger ◽

Dennis R. Claflin ◽

John A. Faulkner

Keyword(s):

Skeletal Muscle ◽

Plasma Membranes ◽

Calcium Influx ◽

Contractile Activity ◽

Muscle Fibers ◽

Mdx Mice ◽

Poloxamer 188 ◽

Influx Pathways ◽

Lumbrical Muscles ◽

Free Environment

Duchenne Muscular Dystrophy is a genetic disease caused by the lack of the protein dystrophin. Dystrophic muscles are highly susceptible to contraction-induced injury, and following contractile activity, have disrupted plasma membranes that allow leakage of calcium ions into muscle fibers. Because of the direct relationship between increased intracellular calcium concentration and muscle dysfunction, therapeutic outcomes may be achieved through the identification and restriction of calcium influx pathways. Our purpose was to determine the contribution of sarcolemmal lesions to the force deficits caused by contraction-induced injury in dystrophic skeletal muscles. Using isolated lumbrical muscles from dystrophic ( mdx) mice, we demonstrate for the first time that poloxamer 188 (P188), a membrane-sealing poloxamer, is effective in reducing the force deficit in a whole mdx skeletal muscle. A reduction in force deficit was also observed in mdx muscles that were exposed to a calcium-free environment. These results, coupled with previous observations of calcium entry into mdx muscle fibers during a similar contraction protocol, support the interpretation that extracellular calcium enters through sarcolemmal lesions and contributes to the force deficit observed in mdx muscles. The results provide a basis for potential therapeutic strategies directed at membrane stabilization of dystrophin-deficient skeletal muscle fibers.

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Regenerating myofibers in tibialis anterior muscles of young ‘MDX’ mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127943 ◽

1987 ◽

Vol 45 ◽

pp. 726-727

Author(s):

H. D. Geissinge ◽

L.D. Rhodes

Keyword(s):

Muscular Dystrophy ◽

Mouse Model ◽

Tibialis Anterior ◽

Physiological Activity ◽

Mdx Mice ◽

Mode Of Inheritance

A recently discovered mouse model (‘mdx’) for muscular dystrophy in man may be of considerable interest, since the disease in ‘mdx’ mice is inherited by the same mode of inheritance (X-linked) as the human Duchenne (DMD) muscular dystrophy. Unlike DMD, which results in a situation in which the continual muscle destruction cannot keep up with abortive regenerative attempts of the musculature, and the sufferers of the disease die early, the disease in ‘mdx’ mice appears to be transient, and the mice do not die as a result of it. In fact, it has been reported that the severely damaged Tibialis anterior (TA) muscles of ‘mdx’ mice seem to display exceptionally good regenerative powers at 4-6 weeks, so much so, that these muscles are able to regenerate spontaneously up to their previous levels of physiological activity.

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Faculty Opinions recommendation of Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725296116.793509475 ◽

2015 ◽

Author(s):

James Tidball

Keyword(s):

Innate Immunity ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Therapeutic Target ◽

Mdx Mice ◽

Toll Like Receptor ◽

Toll Like Receptor 4

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Age-Dependent Dysregulation of Muscle Vasculature and Blood Flow Recovery after Hindlimb Ischemia in the mdx Model of Duchenne Muscular Dystrophy

Biomedicines ◽

10.3390/biomedicines9050481 ◽

2021 ◽

Vol 9 (5) ◽

pp. 481

Author(s):

Paulina Podkalicka ◽

Olga Mucha ◽

Katarzyna Kaziród ◽

Iwona Bronisz-Budzyńska ◽

Sophie Ostrowska-Paton ◽

...

Keyword(s):

Blood Flow ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Blood Vessels ◽

Mdx Mice ◽

Hindlimb Ischemia ◽

Double Positive ◽

Functional Studies ◽

Age Dependent ◽

Dystrophic Mice

Duchenne muscular dystrophy (DMD), caused by a lack of functional dystrophin, is characterized by progressive muscle degeneration. Interestingly, dystrophin is also expressed in endothelial cells (ECs), and insufficient angiogenesis has already been hypothesized to contribute to DMD pathology, however, its status in mdx mice, a model of DMD, is still not fully clear. Our study aimed to reveal angiogenesis-related alterations in skeletal muscles of mdx mice compared to wild-type (WT) counterparts. By investigating 6- and 12-week-old mice, we sought to verify if those changes are age-dependent. We utilized a broad spectrum of methods ranging from gene expression analysis, flow cytometry, and immunofluorescence imaging to determine the level of angiogenic markers and to assess muscle blood vessel abundance. Finally, we implemented the hindlimb ischemia (HLI) model, more biologically relevant in the context of functional studies evaluating angiogenesis/arteriogenesis processes. We demonstrated that both 6- and 12-week-old dystrophic mice exhibited dysregulation of several angiogenic factors, including decreased vascular endothelial growth factor A (VEGF) in different muscle types. Nonetheless, in younger, 6-week-old mdx animals, neither the abundance of CD31+α-SMA+ double-positive blood vessels nor basal blood flow and its restoration after HLI was affected. In 12-week-old mdx mice, although a higher number of CD31+α-SMA+ double-positive blood vessels and an increased percentage of skeletal muscle ECs were found, the abundance of pericytes was diminished, and blood flow was reduced. Moreover, impeded perfusion recovery after HLI associated with a blunted inflammatory and regenerative response was evident in 12-week-old dystrophic mice. Hence, our results reinforce the hypothesis of age-dependent angiogenic dysfunction in dystrophic mice. In conclusion, we suggest that older mdx mice constitute an appropriate model for preclinical studies evaluating the effectiveness of vascular-based therapies aimed at the restoration of functional angiogenesis to mitigate DMD severity.

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Have Duchenne Muscular Dystrophy Patients an Increased Cancer Risk?

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210676 ◽

2021 ◽

pp. 1-5

Author(s):

Gian Luca Vita ◽

Luisa Politano ◽

Angela Berardinelli ◽

Giuseppe Vita

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Systematic Investigation ◽

Mdx Mice ◽

True Incidence ◽

Patients With Cancer ◽

Dmd Gene ◽

Age Range ◽

Prevalence Of Cancer

Background: Increasing evidence suggests that Duchenne muscular dystrophy (DMD) gene is involved in the occurrence of different types of cancer. Moreover, development of sarcomas was reported in mdx mice, the murine model of DMD, in older age. So far, nine isolated DMD patients were reported with concomitant cancer, four of whom with rhabdomyosarcoma (RMS), but no systematic investigation was performed about the true incidence of cancer in DMD. Methods: All members of the Italian Association of Myology were asked about the occurrence of cancer in their DMD patients in the last 30 years. Results: Four DMD patients with cancer were reported after checking 2455 medical records. One developed brain tumour at the age of 35 years. Two patients had alveolar RMS at 14 and 17 years of age. The fourth patient had a benign enchondroma when 11-year-old. Conclusion: Prevalence of cancer in general in the Italian DMD patients does not seem to be different from that in the general population with the same age range. Although the small numbers herein presented do not allow definitive conclusion, the frequent occurrence of RMS in DMD patients raises an alert for basic researchers and clinicians. The role of DMD gene in cancer merits further investigations.

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Autophagy in the heart is enhanced and independent of disease progression in mus musculus dystrophinopathy models

JRSM Cardiovascular Disease ◽

10.1177/2048004019879581 ◽

2019 ◽

Vol 8 ◽

pp. 204800401987958

Author(s):

HR Spaulding ◽

C Ballmann ◽

JC Quindry ◽

MB Hudson ◽

JT Selsby

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Disease Progression ◽

Gene Mutations ◽

Dystrophin Gene ◽

Mdx Mice ◽

Dystrophin Deficiency ◽

Muscle Wasting Disease ◽

Dystrophin Protein

Background Duchenne muscular dystrophy is a muscle wasting disease caused by dystrophin gene mutations resulting in dysfunctional dystrophin protein. Autophagy, a proteolytic process, is impaired in dystrophic skeletal muscle though little is known about the effect of dystrophin deficiency on autophagy in cardiac muscle. We hypothesized that with disease progression autophagy would become increasingly dysfunctional based upon indirect autophagic markers. Methods Markers of autophagy were measured by western blot in 7-week-old and 17-month-old control (C57) and dystrophic (mdx) hearts. Results Counter to our hypothesis, markers of autophagy were similar between groups. Given these surprising results, two independent experiments were conducted using 14-month-old mdx mice or 10-month-old mdx/Utrn± mice, a more severe model of Duchenne muscular dystrophy. Data from these animals suggest increased autophagosome degradation. Conclusion Together these data suggest that autophagy is not impaired in the dystrophic myocardium as it is in dystrophic skeletal muscle and that disease progression and related injury is independent of autophagic dysfunction.

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Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy

International Journal of Molecular Sciences ◽

10.3390/ijms22137063 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7063

Author(s):

Sharon Mordechay ◽

Shaun Smullen ◽

Paul Evans ◽

Olga Genin ◽

Mark Pines ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Motor Coordination ◽

Mdx Mice ◽

Cell Viability Assay ◽

Muscle Fibrosis ◽

Antifibrotic Therapy ◽

Racemic Form ◽

Viability Assay

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(−)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form’s effect. No effect was observed for (−)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (−)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (−)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

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