Reduction in mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised compared with sedentary mdx mice

By proteomic analysis we found an up-regulation of four carbonic anhydrase-3 (CA3) isoforms and a down-regulation of superoxide dismutase [Cu-Zn] (SODC) in quadriceps of sedentary X-linked muscular dystrophy (mdx) mice as compared with wild–type (WT) mice and the levels were significantly restored to WT values following low-intensity endurance exercise.

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Taurine and Methylprednisolone Administration at Close Proximity to the Onset of Muscle Degeneration Is Ineffective at Attenuating Force Loss in the Hind-Limb of 28 Days Mdx Mice

Sports ◽

10.3390/sports6040109 ◽

2018 ◽

Vol 6 (4) ◽

pp. 109 ◽

Cited By ~ 1

Author(s):

Robert Barker ◽

Chris van der Poel ◽

Deanna Horvath ◽

Robyn Murphy

Keyword(s):

Drinking Water ◽

Duchenne Muscular Dystrophy ◽

Hind Limb ◽

Mdx Mouse ◽

Mdx Mice ◽

Muscle Degeneration ◽

Wild Type ◽

Close Proximity ◽

Contractile Characteristics

An increasing number of studies have shown supplementation with the amino acid taurine to have promise in ameliorating dystrophic symptoms in the mdx mouse model of Duchenne Muscular Dystrophy (DMD). Here we build on this limited body of work by investigating the efficacy of supplementing mdx mice with taurine postnatally at a time suggestive of when dystrophic symptoms would begin to manifest in humans, and when treatments would likely begin. Mdx mice were given either taurine (mdx tau), the steroid alpha methylprednisolone (PDN), or tau + PDN (mdx tau + PDN). Taurine (2.5% wt/vol) enriched drinking water was given from 14 days and PDN (1 mg/kg daily) from 18 days. Wild-type (WT, C57BL10/ScSn) mice were used as a control to mdx mice to represent healthy tissue. In the mdx mouse, peak damage occurs at 28 days, and in situ assessment of contractile characteristics showed that taurine, PDN, and the combined taurine + PDN treatment was ineffective at attenuating the force loss experienced by mdx mice. Given the benefits of taurine as well as methylprednisolone reported previously, when supplemented at close proximity to the onset of severity muscle degeneration these benefits are no longer apparent.

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Twenty-one days of low-intensity eccentric training improve morphological characteristics and function of soleus muscles of mdx mice

Scientific Reports ◽

10.1038/s41598-020-79168-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Paulo S. Pedrazzani ◽

Tatiana O. P. Araújo ◽

Emilly Sigoli ◽

Isabella R. da Silva ◽

Daiane Leite da Roza ◽

...

Keyword(s):

Soleus Muscle ◽

Morphological Characteristics ◽

Training Group ◽

Mdx Mice ◽

Eccentric Training ◽

Active Force ◽

Wild Type ◽

Low Intensity ◽

Sedentary Group ◽

Group 3

AbstractDuchene muscular dystrophy (DMD) is caused by the absence of the protein dystrophin, which leads to muscle weakness, progressive degeneration, and eventually death due to respiratory failure. Low-intensity eccentric training (LIET) has been used as a rehabilitation method in skeletal muscles after disuse. Recently, LIET has also been used for rehabilitating dystrophic muscles, but its effects are still unclear. The purpose of this study was to investigate the effects of 21 days of LIET in dystrophic soleus muscle. Thirty-six male mdx mice were randomized into six groups (n = 6/each): mdx sedentary group; mdx training group-3 days; mdx training group-21 days; wild-type sedentary group; wild-type training group-3 days and wild-type training group-21 days. After the training sessions, animals were euthanized, and fragments of soleus muscles were removed for immunofluorescence and histological analyses, and measurements of active force and Ca2+ sensitivity of the contractile apparatus. Muscles of the mdx training group-21 days showed an improvement in morphological characteristics and an increase of active force when compared to the sedentary mdx group. The results show that LIET can improve the functionality of dystrophic soleus muscle in mice.

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Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

Molecular Biology of the Cell ◽

10.1091/mbc.5.10.1159 ◽

1994 ◽

Vol 5 (10) ◽

pp. 1159-1167 ◽

Cited By ~ 17

Author(s):

W F Denetclaw ◽

F W Hopf ◽

G A Cox ◽

J S Chamberlain ◽

R A Steinhardt

Keyword(s):

Single Channel ◽

Calcium Regulation ◽

Full Length ◽

Mdx Mice ◽

Free Calcium ◽

Muscle Degeneration ◽

Channel Activity ◽

Mouse Muscle ◽

Calcium Dependent ◽

Normal Calcium

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

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Regenerative capacity of the dystrophic (mdx) diaphragm after induced injury

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00146.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. R961-R968 ◽

Cited By ~ 21

Author(s):

Stefan Matecki ◽

Ghiabe H. Guibinga ◽

Basil J. Petrof

Keyword(s):

Connective Tissue ◽

Isometric Force ◽

Mdx Mouse ◽

Mdx Mice ◽

Wild Type ◽

Regenerative Capacity ◽

Generating Capacity ◽

Massive Necrosis ◽

Characteristic Features

Duchenne muscular dystrophy is characterized by myofiber necrosis, muscle replacement by connective tissue, and crippling weakness. Although the mdx mouse also lacks dystrophin, most muscles show little myofiber loss or functional impairment. An exception is the mdx diaphragm, which is phenotypically similar to the human disease. Here we tested the hypothesis that the mdx diaphragm has a defective regenerative response to necrotic injury, which could account for its severe phenotype. Massive necrosis was induced in mdx and wild-type (C57BL10) mouse diaphragms in vivo by topical application of notexin, which destroys mature myofibers while leaving myogenic precursor satellite cells intact. At 4 h after acute exposure to notexin, >90% of diaphragm myofibers in both wild-type and mdx mice demonstrated pathological sarcolemmal leakiness, and there was a complete loss of isometric force-generating capacity. Both groups of mice showed strong expression of embryonic myosin within the diaphragm at 5 days, which was largely extinguished by 20 days after injury. At 60 days postinjury, wild-type diaphragms exhibited a persistent loss (∼25%) of isometric force-generating capacity, associated with a trend toward increased connective tissue infiltration. In contrast, mdx diaphragms achieved complete functional recovery of force generation to noninjured values, and there was no increase in muscle connective tissue over baseline. These data argue against any loss of intrinsic regenerative capacity within the mdx diaphragm, despite characteristic features of major dystrophic pathology being present. Our findings support the concept that significant latent regenerative capacity resides within dystrophic muscles, which could potentially be exploited for therapeutic purposes.

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Upregulation of store-operated Ca2+ entry in dystrophic mdx mouse muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00524.2009 ◽

2010 ◽

Vol 299 (1) ◽

pp. C42-C50 ◽

Cited By ~ 59

Author(s):

Joshua N. Edwards ◽

Oliver Friedrich ◽

Tanya R. Cully ◽

Frederic von Wegner ◽

Robyn M. Murphy ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Fibers ◽

Mdx Mouse ◽

Cell Necrosis ◽

Wild Type ◽

Dystrophic Muscle ◽

Adult Skeletal Muscle ◽

Mouse Muscle ◽

Threefold Increase ◽

Tubular System

Store-operated Ca2+ entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca2+ from the transverse tubule into the junctional cleft during periods of depleting Ca2+ release. In dystrophic muscle fibers, SOCE may be a source of Ca2+ overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca2+ release. To examine this, Ca2+ in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca2+ from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca2+ transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca2+ concentrations ([Ca2+]SR). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca2+ into the fiber for an equivalent depletion of [Ca2+]SR via SOCE, it remains unclear whether this is deleterious.

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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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Lengthening-contractions in isolated myocardium impact force development and worsen cardiac contractile function in the mdx mouse model of muscular dystrophy

Journal of Applied Physiology ◽

10.1152/japplphysiol.00253.2010 ◽

2011 ◽

Vol 110 (2) ◽

pp. 512-519 ◽

Cited By ~ 5

Author(s):

Ying Xu ◽

Dawn A. Delfín ◽

Jill A. Rafael-Fortney ◽

Paul M. L. Janssen

Keyword(s):

Contractile Function ◽

Wild Type Mouse ◽

Mdx Mouse ◽

Mdx Mice ◽

Wild Type ◽

Cardiac Contractile Function ◽

Lengthening Contractions ◽

Peak Tension ◽

The Impact

Lengthening-contractions exert eccentric stress on myofibers in normal myocardium. In congestive heart failure caused by a variety of diseases, the impact of lengthening-contractions of myocardium likely becomes more prevalent and severe. The present study introduces a method to investigate the role of stretching imposed by repetitive lengthening-contractions in myocardium under near-physiological conditions. By exerting various stretch-release ramps while the muscle is contracting, consecutive lengthening-contractions and their potential detrimental effect on cardiac function can be studied. We tested our model and hypothesis in age-matched (young and adult) mdx and wild-type mouse right ventricular trabeculae. These linear and ultrathin muscles possess all major cardiac cell types, and their contractile behavior very closely mimics that of the whole myocardium. In the first group of experiments, 10 lengthening-contractions at various magnitudes of stretch were performed in trabeculae from 10-wk-old mdx and wild-type mice. In the second group, 100 lengthening-contractions at various magnitudes were conducted in trabeculae from 10- and 20-wk-old mice. The peak isometric active developed tension (Fdev, in mN/mm2) and kinetic parameters time to peak tension (TTP, in ms) and time from peak tension to half-relaxation (RT50, in ms) were measured. Our results indicate lengthening-contractions significantly impact contractile behavior, and that dystrophin-deficient myocardium in mdx mice is significantly more susceptible to these damaging lengthening-contractions. The results indicate that lengthening-contractions in intact myocardium can be used in vitro to study this emerging contributor to cardiomyopathy.

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Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from mdx mouse muscle

Cellular & Molecular Biology Letters ◽

10.2478/s11658-009-0039-8 ◽

2010 ◽

Vol 15 (1) ◽

Cited By ~ 2

Author(s):

Stéphanie Daval ◽

Chantal Rocher ◽

Yan Cherel ◽

Elisabeth Rumeur

Keyword(s):

Muscular Dystrophy ◽

Core Protein ◽

Surface Membrane ◽

Sodium Dodecyl ◽

Mdx Mouse ◽

Mdx Mice ◽

Mouse Muscle ◽

Glycoprotein Complex ◽

Dystrophin Glycoprotein Complex ◽

Dystrophin Glycoprotein

AbstractThe dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.

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Utrophin deficiency worsens cardiac contractile dysfunction present in dystrophin-deficient mdx mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00448.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. H2373-H2378 ◽

Cited By ~ 74

Author(s):

Paul M. L. Janssen ◽

Nitisha Hiranandani ◽

Tessily A. Mays ◽

Jill A. Rafael-Fortney

Keyword(s):

Clinical Signs ◽

Tissue Level ◽

Mdx Mice ◽

Contractile Dysfunction ◽

Muscle Degeneration ◽

Wild Type ◽

Adrenergic Stimulation ◽

Double Knockout ◽

Cardiac Contractile ◽

Cardiac Contractile Dysfunction

The loss of dystrophin in patients with Duchenne muscular dystrophy (DMD) causes devastating skeletal muscle degeneration and cardiomyopathy. Dystrophin-deficient ( mdx) mice have a much milder phenotype, whereas double knockout (DKO) mice lacking both dystrophin and its homolog, utrophin, exhibit the clinical signs observed in DMD patients. We have previously shown that DKO and mdx mice have similar severities of histological features of cardiomyopathy, but no contractile functional measurements of DKO heart have ever been carried out. To investigate whether DKO mice display cardiac dysfunction at the tissue level, contractile response of the myocardium was tested in small, unbranched, ultrathin, right ventricular muscles. Under near physiological conditions, peak isometric active developed tension (Fdev, in mN/mm2) at a stimulation frequency of 4 Hz was depressed in DKO mice (15.3 ± 3.7, n = 8) compared with mdx mice (24.2 ± 5.4, n = 7), which in turn were depressed compared with wild-type (WT) control mice (33.2 ± 4.5, n = 7). This reduced Fdev was also observed at frequencies within the murine physiological range; at 12 Hz, Fdev was (in mN/mm2) 11.4 ± 1.8 in DKO, 14.5 ± 4.2 in mdx, and 28.8 ± 5.4 in WT mice. The depression of Fdev was observed over the entire frequency range of 4–14 Hz and was significant between DKO versus mdx mice, as well as between DKO or mdx mice versus WT mice. Under β-adrenergic stimulation (1 μmol/l isoproterenol), Fdev in DKO preparations was only (in mN/mm2) 14.7 ± 5.1 compared with 30.9 ± 8.9 in mdx and 41.0 ± 4.9 in WT mice. These data show that cardiac contractile dysfunction of mdx mice is generally worsened in mice also lacking utrophin.

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Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00144.2012 ◽

2012 ◽

Vol 303 (5) ◽

pp. C567-C576 ◽

Cited By ~ 13

Author(s):

Tanya R. Cully ◽

Joshua N. Edwards ◽

Oliver Friedrich ◽

D. George Stephenson ◽

Robyn M. Murphy ◽

...

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Mdx Mouse ◽

Wild Type ◽

Mouse Muscle ◽

Muscle Plasma Membrane ◽

Partner Protein ◽

Close Relationship ◽

The Common ◽

T System

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca2+ from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca2+. The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca2+ entry (SOCE) can also be activated upon release of SR Ca2+. In muscle, the SR Ca2+ sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca2+ channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca2+ influx in mdx compared with WT muscle for similar levels of SR Ca2+ release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca2+ via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca2+ extrusion properties as in the WT muscle.

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