Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements

Skeletal muscle microtubules (MTs) form a nonclassic grid-like network, which has so far been documented in static images only. We have now observed and analyzed dynamics of GFP constructs of MT and Golgi markers in single live fibers and in the whole mouse muscle in vivo. Using confocal, intravital, and superresolution microscopy, we find that muscle MTs are dynamic, growing at the typical speed of ∼9 µm/min, and forming small bundles that build a durable network. We also show that static Golgi elements, associated with the MT-organizing center proteins γ-tubulin and pericentrin, are major sites of muscle MT nucleation, in addition to the previously identified sites (i.e., nuclear membranes). These data give us a framework for understanding how muscle MTs organize and how they contribute to the pathology of muscle diseases such as Duchenne muscular dystrophy.

Download Full-text

Reparative dysfunction during muscle regeneration in Duchenne muscular dystrophy: therapeutic approaches to modulating the muscle environment and muscle stem cells

10.32469/10355/76151 ◽

2019 ◽

Author(s):

◽

Michael Everette Nance

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Regeneration ◽

Genetic Mutation ◽

Critical Determinant ◽

Muscle Stem Cells ◽

Dystrophin Protein

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Duchenne muscular dystrophy (DMD) is a lethal muscular dystrophy resulting from functional loss of the dystrophin protein, a critical sub-sarcolemmal protein involved in membrane stability. While reparative dysfunction is thought to be a critical determinant of disease progression in humans, regeneration is not significantly impaired in the murine muscular dystrophy (mdx) model. Furthermore, it is not well understood if reparative dysfunction is related to inherent defects in stem cells or chronic alterations in the muscle environment due to disease related remodeling. To address these observed discrepancies, we adapted a whole muscle transplant model to study the in vivo regeneration of intact pieces of skeletal muscle from normal and dystrophic dogs (cDMD), a physiological and clinically relevant model to humans. Regeneration in cDMD muscle grafts was significantly attenuated compared to normal and predisposed to the development of skeletal muscle tumors. We used an adeno-associated virus (AAV) expressing a micro-dystrophin protein to specifically rescue the muscle environment by preventing fiber damage while retaining dystrophin-null SCs. AAV.micro-dystrophin rescued the environment by improving fibrosis, stiffness, and fiber orientation, which significantly improved early muscle regeneration but not late regeneration (2 greater than and less than 4 months post-transplant) via enhancing muscle stem cells differentiation. We next developed Cre- and CRISPR-cas9 gene editing strategies to test the ability of AAV serotype 9 to transduce and treat the genetic mutation in muscle stem cells. We observed efficient SC transduction when used as a single vector expressing Cre. Dual-vector CRISPR-cas9 SC transduction was inefficient and likely related to the requirement for two vectors, promoter usage, and mechanistic differences between Cre-recombination and CRISPR genome editing.

Download Full-text

Peptide-Functionalized Dendrimer Nanocarriers for Targeted Microdystrophin Gene Delivery

Pharmaceutics ◽

10.3390/pharmaceutics13122159 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2159

Author(s):

Jessica Hersh ◽

José Manuel Condor Capcha ◽

Camila Iansen Irion ◽

Guerline Lambert ◽

Mauricio Noguera ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Gene Delivery ◽

Nuclear Membrane ◽

Targeted Delivery ◽

Intracellular Transport ◽

Muscle Cells ◽

Skeletal Muscle Cells

Gene therapy is a good alternative for determined congenital disorders; however, there are numerous limitations for gene delivery in vivo including targeted cellular uptake, intracellular trafficking, and transport through the nuclear membrane. Here, a modified G5 polyamidoamine (G5 PAMAM) dendrimer–DNA complex was developed, which will allow cell-specific targeting to skeletal muscle cells and transport the DNA through the intracellular machinery and the nuclear membrane. The G5 PAMAM nanocarrier was modified with a skeletal muscle-targeting peptide (SMTP), a DLC8-binding peptide (DBP) for intracellular transport, and a nuclear localization signaling peptide (NLS) for nuclear uptake, and polyplexed with plasmid DNA containing the GFP-tagged microdystrophin (µDys) gene. The delivery of µDys has been considered as a therapeutic modality for patients suffering from a debilitating Duchenne muscular dystrophy (DMD) disorder. The nanocarrier–peptide–DNA polyplexes were prepared with different charge ratios and characterized for stability, size, surface charge, and cytotoxicity. Using the optimized nanocarrier polyplexes, the transfection efficiency in vitro was determined by demonstrating the expression of the GFP and the µDys protein using fluorescence and Western blotting studies, respectively. Protein expression in vivo was determined by injecting an optimal nanocarrier polyplex formulation to Duchenne model mice, mdx4Cv. Ultimately, these nanocarrier polyplexes will allow targeted delivery of the microdystrophin gene to skeletal muscle cells and result in improved muscle function in Duchenne muscular dystrophy patients.

Download Full-text

miR-486 is an epigenetic modulator of Duchenne muscular dystrophy pathologies

10.1101/2021.06.14.448387 ◽

2021 ◽

Author(s):

Rylie M. Hightower ◽

Adrienne Samani ◽

Andrea Louise Reid ◽

Katherine G English ◽

Michael A Lopez ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Development ◽

Cardiac Fibrosis ◽

Sequencing Data ◽

Microrna Target ◽

Mouse Muscle ◽

Metabolic Defects ◽

Dystrophic Disease

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder resulting in muscle weakness and cardiomyopathy. MicroRNAs have been shown to play essential roles in muscle development, metabolism, and disease pathologies. We demonstrated that miR-486 expression is reduced in DMD muscles and its expression levels correlate with dystrophic disease severity. MicroRNA-486 knockout mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects that were exacerbated on the dystrophic mdx5cv background. We integrated RNA-sequencing and chimeric eCLIP-sequencing data to identify direct in vivo targets of miR-486 and associated dysregulated gene signatures in skeletal muscle. In comparison to our DMD mouse muscle transcriptomes, we identified several of these miR-486 muscle targets including known modulators of dystrophinopathy disease symptoms. Together, our studies identify miR-486 as a driver of muscle remodeling in DMD, a useful biomarker for dystrophic disease progression, and highlight chimeric eCLIP-sequencing as a useful tool to identify direct in vivo microRNA target transcripts.

Download Full-text

Biochemical Changes in Progressive Muscular Dystrophy. IX. Synthesis of Native Myosin, Actin, and Tropomyosin in the Skeletal Muscle of Mouse as a Function of Muscular Dystrophy

Canadian Journal of Biochemistry ◽

10.1139/o72-055 ◽

1972 ◽

Vol 50 (4) ◽

pp. 409-415 ◽

Cited By ~ 16

Author(s):

Uma Srivastava

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Protein Purification ◽

Gel Electrophoresis ◽

Dystrophic Muscle ◽

Mouse Muscle ◽

Progressive Muscular Dystrophy ◽

Dystrophic Mice

The synthesis of native myosin, actin, and tropomyosin in the skeletal muscle of normal and hereditary dystrophic mice was studied with the help of direct counting as well as acrylamide-gel electrophoresis and protein purification procedures.Labelling of the nascent protein indicated that heavier polysomes from the normal muscle were able to incorporate more radioactivity into the protein than the heavier polysomes from the dystrophic muscles. Contrary to this, lighter polysomes in the dystrophic muscle demonstrated higher incorporation as compared to the normal.Results of in vivo and in vitro incorporation as well as those of acrylamide-gel electrophoresis and protein purification procedures indicated that synthesis of myosin decreased in the dystrophic muscle. The synthesis of actin did not show a significant change either in normal or dystrophic muscle, whereas that of tropomyosin increased sharply in the dystrophic mouse muscle.

Download Full-text

Faculty Opinions recommendation of microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice.

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

10.3410/f.717397850.793153110 ◽

2012 ◽

Author(s):

Michael Rudnicki ◽

Alessandra Pasut

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Regeneration ◽

Skeletal Muscle Regeneration

Download Full-text

Enzyme studies of skeletal muscle in mice with hereditary muscular dystrophy

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1963.205.5.897 ◽

1963 ◽

Vol 205 (5) ◽

pp. 897-901 ◽

Cited By ~ 45

Author(s):

Marilyn W. McCaman

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Glutathione Reductase ◽

Enzyme Activities ◽

Lactic Dehydrogenase ◽

Normal Muscle ◽

Dystrophic Muscle ◽

Nerve Section ◽

Mouse Muscle ◽

Dystrophic Mouse

The activities of 20 enzymes in normal, heterozygous, and dystrophic mouse muscle were studied by means of quantitative microchemical methods. Enzyme activities in normal and heterozygous muscle were essentially the same. In dystrophic muscle glucose-6-P dehydrogenase, 6-P-gluconic dehydrogenase, glutathione reductase, peptidase, ß-glucuronidase, and glucokinase activities were significantly higher than in normal muscle, while α-glycero-P dehydrogenase and lactic dehydrogenase activities were significantly lower. The pattern of enzyme activities found in normal gastrocnemius denervated by nerve section was strikingly similar to that in dystrophic muscle.

Download Full-text

In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

Genome Medicine ◽

10.1186/s13073-021-00876-0 ◽

2021 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Menglong Chen ◽

Hui Shi ◽

Shixue Gou ◽

Xiaomin Wang ◽

Lei Li ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Genome Editing ◽

Large Scale ◽

Gene Editing ◽

High Efficiency ◽

Muscle Fibers ◽

Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

Download Full-text

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.

Download Full-text