Circadian Genes as Exploratory Biomarkers in DMD: Results From Both the mdx Mouse Model and Patients

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients’ plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

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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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Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy

Clinical Chemistry ◽

10.1093/clinchem/47.3.451 ◽

2001 ◽

Vol 47 (3) ◽

pp. 451-458 ◽

Cited By ~ 31

Author(s):

Angelika Hammerer-Lercher ◽

Petra Erlacher ◽

Reginald Bittner ◽

Rudolf Korinthenberg ◽

Daniela Skladal ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Western Blot ◽

Blot Analysis ◽

Cardiac Troponin ◽

Western Blot Analysis ◽

Clinical Evidence ◽

Mdx Mouse ◽

Muscle Biopsies

Abstract Background: Because of controversial earlier studies, the purpose of this study was to provide novel experimental and additional clinical data regarding the possible reexpression of cardiac troponin T (cTnT) in regenerating skeletal muscle in Duchenne muscular dystrophy (DMD). Methods: Plasma from 14 patients (mean age, 7.5 years; range, 5.7–19.4 years) with DMD was investigated for creatine kinase (CK), the CK MB isoenzyme (CKMB), cTnT and cardiac troponin I (cTnI), and myoglobin. cTnT concentrations were measured by an ELISA (second-generation assay; Roche) using the ES 300 Analyzer. cTnI, myoglobin, and CKMB were measured by an ELISA using the ACCESS System (Beckman Diagnostics). Troponin isoform expression was studied by Western blot analysis in remnants of skeletal muscle biopsies of three patients with DMD and in an animal model of DMD (mdx mice; n = 6). Results: There was no relation of cTnT and cTnI to clinical evidence for cardiac failure. cTnI concentrations remained below the upper reference limit in all patients. cTnT was increased (median, 0.11 μg/L; range, 0.06–0.16 μg/L) in 50% of patients. The only significant correlation was found for CK (median, 3938 U/L; range, 2763–5030 U/L) with age (median, 7.5 years; range, 6.8–10.9 years; r = −0.762; P = 0.042). Western blot analysis of human or mouse homogenized muscle specimens showed no evidence for cardiac TnT and cTnI expression, despite strong signals for skeletal muscle troponin isoforms. Conclusions: We found no evidence for cTnT reexpression in human early-stage DMD and in mdx mouse skeletal muscle biopsies. Discrepancies of cTnT and cTnI in plasma samples of DMD patients were found, but neither cTnT nor cTnI plasma concentrations were related with other clinical evidence for cardiac involvement.

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Abstract 252: The Capsaicin Sensitive Afferent Neuron Innervating Skeletal Muscle is Abnormal in the Mdx Mouse

Circulation Research ◽

10.1161/res.121.suppl_1.252 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Qinglu Li ◽

Mary Garry

Keyword(s):

Blood Pressure ◽

Skeletal Muscle ◽

Muscular Dystrophy ◽

Blood Pressure Response ◽

Dystrophin Gene ◽

Mdx Mouse ◽

Mdx Mice ◽

Afferent Neuron ◽

Afferent Neurons ◽

Left Common Iliac Artery

Duchenne muscular dystrophy (DMD) is a severe type of muscular dystrophy caused by a mutation of the dystrophin gene at locus Xp21, located on the short arm of the X chromosome. Muscle wasting and weakness are common in DMD and in the murine mdx model. We previously demonstrated Group III and IV afferent neurons, which innervate skeletal muscle and control blood pressure and heart rate in response to exercise, are abnormal in settings of ischemia and atrophy; such as cardiomyopathy. We hypothesized that these afferent neurons would also display abnormalities in the mdx mouse. To test this hypothesis, we developed a decerebrate mouse model using 10 wk and 6 mo old male BL10 WT and MDX mice to test mean arterial pressure (MAP) responses to intra-arterial capsaicin (IA-Cap; a specific stimulant of group IV afferent neurons). Mice were anesthetized and MAP was continuously recorded with a pressure transducer in the left carotid artery after which the animal was rendered decerebrate. Following decerebration, anesthesia was discontinued and IA-Cap (0,003-1ug/100ul) was delivered via the left common iliac artery. In rats, we have demonstrated this to be a valid model for evaluating MAP responses to activation of metabolically active afferent neurons. We observed that MAP increased in a dose-related fashion in both 10wk and 6 mo old WT and MDX, while 10 wk old MDX mouse had a normal response, the 6 months MDX mouse response was significantly blunted when compared to WT. To test whether these abnormalities are related to the onset of cardiomyopathy, Echocardiography was performed using 6 months old BL10 WT and MDX mice, no abnormality was found in terms of LV dimensions and function in MDX mice comparing with WT mice. Further studies will be performed to determine whether these abnormalities are inherent to changes in the skeletal muscle of the mdx mouse. We conclude that this murine model displays pressor responses to IA-Cap, similar to the rat and that MDX mice have a blunted blood pressure response to IA-Cap. These results indicate that abnormalities exist within the skeletal muscle afferent neurons in the mdx model.

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Metabolomic Analyses Reveal Extensive Progenitor Cell Deficiencies in a Mouse Model of Duchenne Muscular Dystrophy

Metabolites ◽

10.3390/metabo8040061 ◽

2018 ◽

Vol 8 (4) ◽

pp. 61 ◽

Cited By ~ 10

Author(s):

Josiane Joseph ◽

Dong Cho ◽

Jason Doles

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Progenitor Cell ◽

Treatment Options ◽

Cell Types ◽

Mdx Mouse ◽

Mdx Mice ◽

Metabolic Alterations ◽

Potential Contributor

Duchenne muscular dystrophy (DMD) is a musculoskeletal disorder that causes severe morbidity and reduced lifespan. Individuals with DMD have an X-linked mutation that impairs their ability to produce functional dystrophin protein in muscle. No cure exists for this disease and the few therapies that are available do not dramatically delay disease progression. Thus, there is a need to better understand the mechanisms underlying DMD which may ultimately lead to improved treatment options. The muscular dystrophy (MDX) mouse model is frequently used to explore DMD disease traits. Though some studies of metabolism in dystrophic mice exist, few have characterized metabolic profiles of supporting cells in the diseased environment. Using nontargeted metabolomics we characterized metabolic alterations in muscle satellite cells (SCs) and serum of MDX mice. Additionally, live-cell imaging revealed MDX-derived adipose progenitor cell (APC) defects. Finally, metabolomic studies revealed a striking elevation of acylcarnitines in MDX APCs, which we show can inhibit APC proliferation. Together, these studies highlight widespread metabolic alterations in multiple progenitor cell types and serum from MDX mice and implicate dystrophy-associated metabolite imbalances in APCs as a potential contributor to adipose tissue disequilibrium in DMD.

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Talin, vinculin and DRP (utrophin) concentrations are increased at mdx myotendinous junctions following onset of necrosis

Journal of Cell Science ◽

10.1242/jcs.107.6.1477 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1477-1483 ◽

Cited By ~ 1

Author(s):

D.J. Law ◽

D.L. Allen ◽

J.G. Tidball

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Cytoskeletal Proteins ◽

Mdx Mouse ◽

Mdx Mice ◽

Myotendinous Junction ◽

Glycoprotein Complex ◽

Transmembrane Glycoprotein ◽

Muscle Necrosis ◽

Myotendinous Junctions

Duchenne muscular dystrophy (DMD) and the myopathy seen in the mdx mouse both result from absence of the protein dystrophin. Structural similarities between dystrophin and other cytoskeletal proteins, its enrichment at myotendinous junctions, and its indirect association with laminin mediated by a transmembrane glycoprotein complex suggest that one of dystrophin's functions in normal muscle is to form one of the links between the actin cytoskeleton and the extracellular matrix. Unlike Duchenne muscular dystrophy patients, mdx mice suffer only transient muscle necrosis, and are able to regenerate damaged muscle tissue. The present study tests the hypothesis that mdx mice partially compensate for dystrophin's absence by upregulating one or more dystrophin-independent mechanisms of cytoskeleton-membrane association. Quantitative analysis of immunoblots of adult mdx muscle samples showed an increase of approximately 200% for vinculin and talin, cytoskeletal proteins that mediate thin filament-membrane interactions at myotendinous junctions. Blots also showed an increase (143%) in the dystrophin-related protein called utrophin, another myotendinous junction constituent, which may be able to substitute for dystrophin directly. Muscle samples from 2-week-old animals, a period immediately preceding the onset of muscle necrosis, showed no significant differences in protein concentration between mdx and controls. Quantitative analyses of confocal images of myotendinous junctions from mdx and control muscles show significantly higher concentrations of talin and vinculin at the myotendinous junctions of mdx muscle. These findings indicate that mdx mice may compensate in part for the absence of dystrophin by increased expression of other molecules that subsume dystrophin's mechanical function.

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AAV micro-dystrophin gene therapy alleviates stress-induced cardiac death but not myocardial fibrosis in >21-m-old mdx mice, an end-stage model of Duchenne muscular dystrophy cardiomyopathy

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2012.05.002 ◽

2012 ◽

Vol 53 (2) ◽

pp. 217-222 ◽

Cited By ~ 44

Author(s):

Brian Bostick ◽

Jin-Hong Shin ◽

Yongping Yue ◽

Nalinda B. Wasala ◽

Yi Lai ◽

...

Keyword(s):

Gene Therapy ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Myocardial Fibrosis ◽

Cardiac Death ◽

Dystrophin Gene ◽

Mdx Mice ◽

Stage Model ◽

End Stage ◽

Duchenne Muscular Dystrophy Cardiomyopathy

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Spectrum of dystrophin gene mutations observed in patients suspected of Duchenne muscular dystrophy in Pakistan

Clinical Medicine ◽

10.7861/clinmedicine.19-3s-s32 ◽

2019 ◽

Vol 19 (Suppl 3) ◽

pp. s32-s32

Author(s):

Zeeshan Ahmed

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Gene Mutations ◽

Dystrophin Gene

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BETs inhibition attenuates oxidative stress and preserves muscle integrity in Duchenne muscular dystrophy

Nature Communications ◽

10.1038/s41467-020-19839-x ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Marco Segatto ◽

Roberta Szokoll ◽

Raffaella Fittipaldi ◽

Cinzia Bottino ◽

Lorenzo Nevi ◽

...

Keyword(s):

Oxidative Stress ◽

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mdx Mouse ◽

Early Event ◽

Dystrophic Muscle ◽

Protein Levels ◽

Bet Inhibitors ◽

Ros Metabolism

AbstractDuchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD.

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MDX mouse myopathy I: Presence or absence of sarcolemmal lesions in tibialis anterior muscles from mice of different ages

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157334 ◽

1989 ◽

Vol 47 ◽

pp. 1070-1071

Author(s):

H.D. Geissinger ◽

L.D. Rhodes

Keyword(s):

Animal Model ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Tibialis Anterior ◽

Extensor Digitorum Longus ◽

Morphological Analysis ◽

Control Mouse ◽

Mdx Mouse ◽

Mdx Mice ◽

Recent Interest

Since the ‘mdx’ mouse appears to have the same basic defect as sufferers of human Duchenne Muscular Dystrophy (DMD), much recent interest in this possible animal model for the human disease has been generated. Perforations in the sarcolemma have been reported recently in the necrotic tibialis anterior (TA) of 35-days-old and the extensor digitorum longus muscles of 39-days-old ‘mdx’ mice. It is the purpose of this communication to find out if these lesions occur not only in necrotic, but also in unaffected, or in centronucleated fibers of the TA of mice which are younger than 35, or older than 39 days.METHODS: TA from 22-, 25-, 41-, 61- and 99-days-old C57BL/10ScSn/MDX and C57BL/lOScSn control mice were pinned on corkboard in a relaxed state, prefixed for 30 minutes in 2.5% glutaraldehyde followed by routine processing for TEM. Appropriate micrographs were evaluated for a more detailed morphological analysis of the sarcolemma (SL) and the basal lamina (BL).RESULTS: It should be stated beforehand that in all muscles examined the BL appeared to be intact. In the muscles of a 25-days-old control mouse the SL appeared quite intact (FIG. 1). In contrast to this small perforations or large tears in the SL could be seen in otherwise unaffected muscles of 22- (FIG. 2), 25- and 41-days-old ‘mdx’ mice, as well as in necrotic and regenerating fibers of mice from these ages.

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