Minimal Consequences of CMAH and DBA/2J Background on a FKRP Deficient Model

Background: Muscular dystrophies (MD) are a large group of genetic diseases characterized by a progressive loss of muscle. The Latent TGFβ Binding Protein 4 (LTBP4) in the DBA/2 background and the Cytidine Monophosphate-sialic Acid Hydroxylase (CMAH) proteins were previously identified as genetic modifiers in severe MD. Objective: We investigated whether these modifiers could also influence a mild phenotype such as the one observed in a mouse model of Limb-Girdle MD2I (LGMD2I). Methods: The FKRPL276I mouse model was backcrossed onto the DBA/2 background, and in separate experiments the Cmah gene was inactivated in FKRPL276I mice by crossing with a Cmah-/- mouse and selecting the double-mutants. The mdx mouse was used as control for these two genome modifications. Consequences at the histological level as well as quantification of expression level by RT-qPCR of genes relevant for muscular dystrophy were then performed. Results: We observed minimal to no effect of the DBA/2 background on the mild FKRPL276I mouse phenotype, while this same background was previously shown to increase inflammation and fibrosis in the mdx mouse. Similarly, the Cmah-/- deletion had no observable effect on the FKRPL276I mouse phenotype whereas it was seen to increase features of regeneration in mdx mice. Conclusions: These modifiers were not observed to impact the severity of the presentation of the mild FKRPL276I model. An interesting association of the CMAH modifier with the regeneration process in the mdx model was seen and sheds new light on the influence of this protein on the dystrophic phenotype.

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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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Evaluation of micro-dystrophin based and dystrophin-independent AAV gene therapies for Duchenne Muscular Dystrophy

10.32469/10355/86465 ◽

2020 ◽

Author(s):

◽

Lakmini P. Wasala

Keyword(s):

Muscular Dystrophy ◽

Mouse Model ◽

Left Ventricular ◽

Upstream Region ◽

Mdx Mouse ◽

Mdx Mice ◽

Wild Type ◽

Ww Domain ◽

Gene Therapies ◽

Complete Deletion

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI-COLUMBIA AT REQUEST OF AUTHOR.] Duchenne Muscular dystrophy (DMD) is the most common, progressive childhood muscular dystrophy with an X-linked inheritance. The major cause of the disease is the mutations in the dystrophin gene which results in the absence of a functional dystrophin protein. Currently there is no permanent cure for DMD. Many genetic and pharmacological approaches have resulted in tremendous improvements in animal models and advanced the mission of finding a permanent cure for DMD. Adeno associated virus (AAV) mediated micro-dystrophin gene therapy is the most promising approach to treat patients irrespective of their type of mutations. Dystrophin independent AAV gene therapies have also shown encouraging data in animal models in subsiding DMD pathology. In engineering micro-dystrophins, it is important to include the most essential regions or domains to achieve maximum benefits, that fits into the AAV. Our goal was to understand the impact of hinge 1 (H1) and hinge 4 (H4) regions in the function of a micro-dystrophin ([micro]Dys) construct. Two novel micro-dystrophins were engineered by complete deletion of either hinge 1 or hinge 4 and packaged in AAV9. Three separate groups of 3-month old male mdx4cv mice tibialis anterior muscles were injected with each novel AAV.[micro]Dys vector and parent vector separately. Three months post injection TA muscle contractile properties were evaluated. Hinge 1 deletion was tolerated by parent [micro]Dys although deletion of hinge 4 reduced the functional performance. Hinge domains played an important part in localization of [micro]Dys to the sarcolemma. Deletion of hinge 1 did not interfere with normal sarcolemmal localization whereas complete deletion of hinge 4 failed to localize [micro]Dys. Both novel [micro]Dys were able to restore dystrophin associated glycoprotein complex (DGC) proteins to the sarcolemma in dystrophin positive fibers. To further analyze which region of hinge 4 that could be devoid of [micro]Dys, we engineered additional four novel [micro]Dys with modifications in only the hinge 4 region, while hinge 1 is intact. Deletion of the region upstream of WW domain was shown to enhance the [micro]Dys function, and any other deletion reduced the performance of [micro]Dys. We also found that deletion of upstream region of WW domain did not interfere in [micro]Dys localization to sarcolemma and other deletions failed to fully restore [micro]Dys to sarcolemma. Next, we developed another micro-dystrophin that combined complete deletion of hinge 1 with deletion of the upstream region of WW domain. This latest [micro]Dys showed to preserve the muscle tetanic force similar to parent [micro]Dys. This is the first study of in-depth evaluation of the importance of the presence or absence of hinge 1 and hinge 4 in the functional performance of micro-dystrophin. These data provide valuable insights in engineering novel micro-dystrophins. One of the major cellular networks affected in DMD is the mitochondrial function and subsequent metabolic homeostasis. PGC-1a is a key transcriptional co-activator of mitochondrial biogenesis and oxidative metabolism in muscle. PGC-1a has previously studied in improving skeletal muscle pathology in mdx mouse model although its therapeutic effects on mdx cardiac pathology has not been evaluated. We delivered AAV9.PGC-1a vector systemically via the tail vein of 12-month old female mdx mice and 4-months post injected we evaluated the left ventricular hemodynamic parameters. AAV.PGC-1a treated mice showed normalization of several left ventricular hemodynamic parameters to the wild type level. Pathway protein analysis revealed overexpression of PGC-1a, resulted in the increased expression of several major transcription factors in oxidative phosphorylation, mitochondrial biogenesis, fatty acid metabolism, electron transport chain. This is the first study to report that cardiac hemodynamic improvements in 4-month treatment of AAV.PGC-1a in aged mdx mice. This study also shows that without replacing dystrophin, PGC-1a overexpression alone resulted in improving cardiac performance by improving cardiac metabolism in mdx mice. The data provided useful insights developing novel therapies in improving DMD cardiomyopathy. In the final study we used another novel isoform of PGC-1a family, PGC-1a4 which has shown to be expressed during resistance training and regulates muscle hypertrophy. As muscle hypertrophy induction has previously shown to be therapeutically effective in mdx mouse model, we delivered AAV.PGC-1a4 systemically and as intramuscular injections. In the mdx4cv mouse model, we could not overexpress the PGC-1a4 protein above the endogenous levels and no cardiac or skeletal muscle function was improved. Although intramuscular delivery of AAV.PGC-1a4 in wild type mice showed overexpression of PGC-1a4 protein above endogenous levels. Wild type mice showed improvements in eccentric force, although muscle cross sectional area or muscle weight did not reach statistical significance. Our study concluded that PGC-1a4 is not a suitable candidate for AAV gene therapy for DMD. In summary, this dissertation provides important discoveries related to development of next-generation micro-dystrophin vectors and dystrophin-independent AAV gene therapies.

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Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy

International Journal of Molecular Sciences ◽

10.3390/ijms23020958 ◽

2022 ◽

Vol 23 (2) ◽

pp. 958

Author(s):

Marco Ponzetti ◽

Argia Ucci ◽

Antonio Maurizi ◽

Luca Giacchi ◽

Anna Teti ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Bone Loss ◽

Mouse Model ◽

Mononuclear Cells ◽

Serum Levels ◽

Mdx Mouse ◽

Mdx Mice ◽

Muscle Fibres ◽

Lipocalin 2

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2−/− mice (MDXxLcn2−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

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Hematopoietic Prostaglandin D Synthase Inhibitor PK007 Decreases Muscle Necrosis in DMD mdx Model Mice

Life ◽

10.3390/life11090994 ◽

2021 ◽

Vol 11 (9) ◽

pp. 994

Author(s):

Sai Yarlagadda ◽

Christina Kulis ◽

Peter G. Noakes ◽

Mark L. Smythe

Keyword(s):

Mouse Model ◽

Creatine Kinase Activity ◽

Mdx Mouse ◽

Mdx Mice ◽

Prostaglandin D2 ◽

Muscle Creatine Kinase ◽

Progressive Muscle Weakness ◽

Prostaglandin D Synthase ◽

Muscle Necrosis ◽

Synthase Inhibitor

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and wasting due to the lack of dystrophin protein. The acute phase of DMD is characterized by muscle necrosis and increased levels of the pro-inflammatory mediator, prostaglandin D2 (PGD2). Inhibiting the production of PGD2 by inhibiting hematopoietic prostaglandin D synthase (HPGDS) may alleviate inflammation and decrease muscle necrosis. We tested our novel HPGDS inhibitor, PK007, in the mdx mouse model of DMD. Our results show that hindlimb grip strength was two-fold greater in the PK007-treated mdx group, compared to untreated mdx mice, and displayed similar muscle strength to strain control mice (C57BL/10ScSn). Histological analyses showed a decreased percentage of regenerating muscle fibers (~20% less) in tibialis anterior (TA) and gastrocnemius muscles and reduced fibrosis in the TA muscle in PK007-treated mice. Lastly, we confirmed that the DMD blood biomarker, muscle creatine kinase activity, was also reduced by ~50% in PK007-treated mdx mice. We conclude that our HPGDS inhibitor, PK007, has effectively reduced muscle inflammation and fibrosis in a DMD mdx mouse model.

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SERCA-mediated calcium uptake in the DBA/2J vs C57BL/10 mdx models of Duchenne muscular dystrophy

10.1101/2021.10.25.465805 ◽

2021 ◽

Author(s):

Riley EG Cleverdon ◽

Kennedy C Whitley ◽

Daniel M Marko ◽

Sophie I Hamstra ◽

Jessica L Braun ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Duchenne Muscular Dystrophy ◽

Sarcoplasmic Reticulum ◽

Muscular Dystrophy ◽

Calcium Uptake ◽

Late Onset ◽

Mdx Mouse ◽

Mdx Mice ◽

Mild Phenotype ◽

Serca Pump

The C57BL/10ScSn-Dmdmdx/J (C57 mdx) mouse is the most commonly used murine model of Duchenne muscular dystrophy (DMD) but displays a mild phenotype with a late onset, greatly limiting translatability to clinical research. In consequence, the D2.B10-Dmdmdx/J (D2 mdx) mouse was created and produces a more severe, early onset phenotype. Mechanistic insights of the D2 mdx phenotype have yet to be elucidated, specifically related to sarcoplasmic reticulum (SR) calcium (Ca2+) handling. In our study, we aimed to determine if SR Ca2+ handling differences in the D2 mdx versus the C57 mdx mouse could explain model phenotypes. Firstly, analyses determined that D2 mdx mice ambulate less and have weaker muscles, but have greater energy expenditure than C57 counterparts. SR Ca2+ handling measures determined that only D2 mdx mice have impaired SR calcium intake in the gastrocnemius, left ventricle and diaphragm. This was coupled with decrements in maximal sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity and greater activation of the Ca2+-activated protease, calpain, in the gastrocnemius. Overall, our study is the first to determine that SR Ca2+ handling is impaired in the D2 mdx mouse, specifically at the level of the SERCA pump.

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Pax7, Pax3 and Mamstr genes are involved in skeletal muscle impaired regeneration of dy2J/dy2J mouse model of Lama2-CMD

Human Molecular Genetics ◽

10.1093/hmg/ddz180 ◽

2019 ◽

Vol 28 (20) ◽

pp. 3369-3390 ◽

Cited By ~ 2

Author(s):

Nurit Yanay ◽

Moran Elbaz ◽

Jenya Konikov-Rozenman ◽

Sharona Elgavish ◽

Yuval Nevo ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Mouse Model ◽

Pathway Analysis ◽

Epithelial Mesenchymal Transition ◽

Congenital Muscular Dystrophy ◽

Mdx Mouse ◽

Mdx Mice ◽

Rna Seq ◽

Mesenchymal Transition

Abstract Congenital muscular dystrophy type-1A (Lama2-CMD) and Duchenne muscular dystrophy (DMD) result from deficiencies of laminin-α2 and dystrophin proteins, respectively. Although both proteins strengthen the sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA-deep sequencing (RNA-Seq) of dy2J/dy2J, Lama2-CMD mouse model, skeletal muscle at 8 weeks of age to elucidate disease pathophysiology. This study is the first report of dy2J/dy2J model whole transcriptome profile. RNA-Seq of the mdx mouse model of DMD and wild-type (WT) mouse was carried as well in order to enable a novel comparison of dy2J/dy2J to mdx. A large group of shared differentially expressed genes (DEGs) was found in dy2J/dy2J and mdx models (1834 common DEGs, false discovery rate [FDR] < 0.05). Enrichment pathway analysis using ingenuity pathway analysis showed enrichment of inflammation, fibrosis, cellular movement, migration and proliferation of cells, apoptosis and necrosis in both mouse models (P-values 3E-10–9E-37). Via canonical pathway analysis, actin cytoskeleton, integrin, integrin-linked kinase, NF-kB, renin–angiotensin, epithelial–mesenchymal transition, and calcium signaling were also enriched and upregulated in both models (FDR < 0.05). Interestingly, significant downregulation of Pax7 was detected in dy2J/dy2J compared to upregulation of this key regeneration gene in mdx mice. Pax3 and Mamstr genes were also downregulated in dy2J/dy2J compared to WT mice. These results may explain the distinct disease course and severity in these models. While the mdx model at that stage shows massive regeneration, the dy2J/dy2J shows progressive dystrophic process. Our data deepen our understanding of the molecular pathophysiology and suggest new targets for additional therapies to upregulate regeneration in Lama2-CMD.

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Fine structure of early degenerating myofibers in the tibialis anterior of young ‘MDX’ mouse

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010010367x ◽

1988 ◽

Vol 46 ◽

pp. 322-323

Author(s):

H.D. Geissinger ◽

C.K. McDonald-Taylor

Keyword(s):

Fine Structure ◽

Muscle Regeneration ◽

Tibialis Anterior ◽

Genetic Defect ◽

Mdx Mouse ◽

Mdx Mice ◽

Histopathological Changes ◽

Electron Microscopic ◽

Dystrophic Mouse ◽

Preliminary Study

A new strain of mice, which had arisen by mutation from a dystrophic mouse colony was designated ‘mdx’, because the genetic defect, which manifests itself in brief periods of muscle destruction followed by episodes of muscle regeneration appears to be X-linked. Further studies of histopathological changes in muscle from ‘mdx’ mice at the light microscopic or electron microscopic levels have been published, but only one preliminary study has been on the tibialis anterior (TA) of ‘mdx’ mice less than four weeks old. Lesions in the ‘mdx’ mice vary between different muscles, and centronucleation of fibers in all muscles studied so far appears to be especially prominent in older mice. Lesions in young ‘mdx’ mice have not been studied extensively, and the results appear to be at variance with one another. The degenerative and regenerative aspects of the lesions in the TA of 23 to 26-day-old ‘mdx’ mice appear to vary quantitatively.

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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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Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice

AJP Cell Physiology ◽

10.1152/ajpcell.00112.2002 ◽

2002 ◽

Vol 283 (3) ◽

pp. C773-C784 ◽

Cited By ~ 41

Author(s):

Karl Rouger ◽

Martine Le Cunff ◽

Marja Steenman ◽

Marie-Claude Potier ◽

Nathalie Gibelin ◽

...

Keyword(s):

Dystrophin Gene ◽

Diaphragm Muscle ◽

Mdx Mouse ◽

Mdx Mice ◽

First Year ◽

Temporal Gene Expression ◽

Cell Functions ◽

Hindlimb Muscles ◽

Genes Encoding ◽

Dystrophin Protein

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.

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