scholarly journals Sarcoplasmic reticulum Ca2+ permeation explored from the lumen side in mdx muscle fibers under voltage control

2012 ◽  
Vol 139 (3) ◽  
pp. 209-218 ◽  
Author(s):  
Gaëlle Robin ◽  
Christine Berthier ◽  
Bruno Allard
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Sarcoplasmic Reticulum ◽  
Muscular Dystrophy ◽  
Action Potentials ◽  
Muscle Fibers ◽  
Cyclopiazonic Acid ◽  
Recovery Phase ◽  
Resting Potential ◽  
Mdx Mice

Under resting conditions, external Ca2+ is known to enter skeletal muscle cells, whereas Ca2+ stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca2+ entry and in the SR Ca2+ leak is still a matter of debate, but several lines of evidence suggest that these Ca2+ fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca2+ dye Fura2, we first demonstrated that the rate of Ca2+ increase in response to cyclopiazonic acid (CPA)–induced inhibition of SR Ca2+-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca2+ leak. However, removal of external Ca2+ reduced the rate of CPA-induced Ca2+ increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca2+ influx in mdx fibers. Fibers were then loaded with the low-affinity Ca2+ dye Fluo5N-AM to measure intraluminal SR Ca2+ changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca2+ changes. Voltage dependence and magnitude of depolarization-induced SR Ca2+ depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca2+ reuptake activity in mdx fibers. Overall, CPA-induced SR Ca2+ leak at −80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca2+ in control fibers. The elevated passive SR Ca2+ leak may contribute to alteration of Ca2+ homeostasis in mdx muscle.

scholarly journals Animal Models for Muscular Dystrophy Show Different Patterns of Sarcolemmal Disruption

1997 ◽  
Vol 139 (2) ◽  
pp. 375-385 ◽  
Author(s):  
Volker Straub ◽  
Jill A. Rafael ◽  
Jeffrey S. Chamberlain ◽  
Kevin P. Campbell
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Animal Models ◽  
Transgenic Mice ◽  
Evans Blue ◽  
Muscle Fibers ◽  
Congenital Muscular Dystrophy ◽  
Mdx Mice ◽  
Skeletal Muscle Fibers

Genetic defects in a number of components of the dystrophin–glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy2J/dy2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin α2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.

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

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.

Duchenne muscular dystrophy gene therapy in the canine model

2018 ◽  
Author(s):  
◽  
Kasun Kodippili
Keyword(s):  
Skeletal Muscle ◽  
Gene Therapy ◽  
Animal Model ◽  
Duchenne Muscular Dystrophy ◽  
Sarcoplasmic Reticulum ◽  
Muscular Dystrophy ◽  
Canine Model ◽  
Disease Pathogenesis ◽  
Preclinical Treatment ◽  
Expression And Activity

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by loss of function mutations in the dystrophin gene, resulting in the absence of dystrophin, a structural protein in muscle. DMD is the most common form of inherited muscle disease in childhood, with an incidence of 1 in 5000 live male births worldwide. The dystrophin-null mdx mouse has been the most widely used animal model for DMD research over the last 30 years. Dystrophin-deficient DMD dogs have also gained prominence as a highly relevant preclinical animal model due to their high phenotypic homology to human DMD patients. Preclinical treatment studies in these dogs are expected to better inform and guide clinical trials in human patients. However, there are still significant gaps in our understanding of the disease pathogenesis and gene therapy in the canine model. The goals of my dissertation work were to establish reagents and methodologies to study preclinical treatment in the canine model, and subsequently characterize the disease pathogenesis and gene therapy in DMD dogs. To this end, I first characterized 65 epitope-specific human dystrophin monoclonal antibodies for their reactivity in canine skeletal and cardiac muscle by both immunofluorescence (IF) staining and western blot. I found species-specific, tissue-specific and assay-specific patterns of reactivity in these antibodies. Importantly, out of the 65 antibodies that I characterized, I recognized 15 antibodies that worked well for canine tissue on both IF staining and western blot, which are recommended for DMD research in the canine model. ... Dystrophin-independent gene therapy for DMD takes advantage of disease-modifying genes that are either structural and/or functional homologues of dystrophin, or alternative targets that are involved in disease pathogenesis. One such alternative target gene is the sarcoplasmic reticulum calcium ATPase 2a (SERCA2a), a pump that transports calcium ions from the cytoplasm into the sarcoplasmic reticulum. I show that SERCA2a expression and activity are impaired, and that calcium homeostasis is dysregulated in DMD dog skeletal muscle. Furthermore, gene therapy with human SERCA2a restored expression and activity of the pump, and improved several aspects of muscle function and histopathology in DMD dog skeletal muscle. In summary, this dissertation work advances our knowledge of the disease pathogenesis and gene therapy prospects in the canine model of DMD, a highly relevant and valuable preclinical DMD model.

Abstract 478: Connexin-43 Reduction Rescues Cardiac and Skeletal Muscle Pathology in a Novel Mouse Model of Duchenne Muscular Dystrophy Symptomatic Carriers

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Julie Nouet ◽  
Eric Himelman ◽  
Diego Fraidenraich
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Muscle Fiber ◽  
Connexin 43 ◽  
Muscle Fibers ◽  
Skeletal Muscle Fiber ◽  
Muscle Pathology ◽  
Female Carriers

Duchenne muscular dystrophy (DMD) and its associated cardiomyopathy manifest in 8-10% of all female carriers however research remains male-centric. Although underrepresented, symptomatic females face the risk of cardiac, respiratory, and skeletal muscle problems. Basic research and clinical trials exclude female carriers therefore developments in treatment expose females to unknown safety and efficacy issues. The bottleneck is largely due to the absence of a faithful mouse model. To generate a mouse model, we injected mdx embryonic stem cells (ESCs) into wild-type (WT) blastocysts ( mdx /WT chimera). The cardiac and skeletal muscle phenotype recapitulates the same generated as a consequence of x-inactivation in human manifesting female patients. In the heart, mdx /WT chimeras develop fibrotic cardiomyopathy. In the skeletal muscle, we found evidence of fibrosis, inflammation and muscle weakness. We found that Connexin-43 (Cx43), the primary gap junctional protein in the heart, was pathologically enhanced and remodeled in mdx /WT chimeras. Cx43 was also enhanced in the dystrophic skeletal muscle. Genetic reduction of Cx43-copy number protected mdx /WT chimeras from cardiac and skeletal muscle fiber damage. The latter result was unexpected because Cx43 is not expressed in mature muscle fibers. Upon further investigation, Cx43 was localized to the mononuclear cells invading the interstitial space between dystrophic skeletal muscle fibers. Pathologically enhanced activity of Cx43 in mdx FACS-macrophages was observed via ethidium bromide uptake and the Cx43 hemichannel peptide mimetic, Gap19, inhibited Cx43 function in a dose-dependent manner. Because an excess of Cx43 has been associated with cell death, we believe that Cx43 reduction in invading mdx macrophages benefits the skeletal muscle of understudied DMD carriers, perhaps by a paracrine mechanism involving macrophage-skeletal muscle fiber communication.

scholarly journals Nutraceutical and pharmaceutical cocktails did not improve muscle function or reduce histological damage in D2-mdx mice

2019 ◽  
Vol 127 (4) ◽  
pp. 1058-1066
Author(s):  
Hannah R. Spaulding ◽  
Tiffany Quindry ◽  
Kayleen Hammer ◽  
John C. Quindry ◽  
Joshua T. Selsby
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Muscle Function ◽  
Sirtuin 1 ◽  
Mdx Mice ◽  
Limb Muscle ◽  
Nicotinamide Riboside ◽  
Histological Damage ◽  
Dystrophin Deficiency

Progressive muscle injury and weakness are hallmarks of Duchenne muscular dystrophy. We showed previously that quercetin (Q) partially protected dystrophic limb muscles from disease-related injury. As quercetin activates PGC-1α through Sirtuin-1, an NAD+-dependent deacetylase, the depleted NAD+ in dystrophic skeletal muscle may limit quercetin efficacy; hence, supplementation with the NAD+ donor, nicotinamide riboside (NR), may facilitate quercetin efficacy. Lisinopril (Lis) protects skeletal muscle and improves cardiac function in dystrophin-deficient mice; therefore, it was included in this study to evaluate the effects of lisinopril used with quercetin and NR. Our purpose was to determine the extent to which Q, NR, and Lis decreased dystrophic injury. We hypothesized that Q, NR, or Lis alone would improve muscle function and decrease histological injury and when used in combination would have additive effects. Muscle function of 11-mo-old DBA (healthy), D2-mdx (dystrophin-deficient), and D2-mdx mice was assessed after treatment with Q, NR, and/or Lis for 7 mo. To mimic typical pharmacology of patients with Duchenne muscular dystrophy, a group was treated with prednisolone (Pred) in combination with Q, NR, and Lis. At 11 mo of age, dystrophin deficiency decreased specific tension and tetanic force in the soleus and extensor digitorum longus muscles and was not corrected by any treatment. Dystrophic muscle was more sensitive to contraction-induced injury, which was partially offset in the QNRLisPred group, whereas fatigue was similar between all groups. Treatments did not decrease histological damage. These data suggest that treatment with Q, NR, Lis, and Pred failed to adequately maintain dystrophic limb muscle function or decrease histological damage. NEW & NOTEWORTHY Despite a compelling rationale and previous evidence to the contrary in short-term investigations, quercetin, nicotinamide riboside, or Lisinopril, alone or in combination, failed to restore muscle function or decrease histological injury in dystrophic limb muscle from D2-mdx mice after long-term administration. Importantly, we also found that in the D2-mdx model, an emerging and relatively understudied model of Duchenne muscular dystrophy dystrophin deficiency caused profound muscle dysfunction and histopathology in skeletal muscle.

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

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. 

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

2021 ◽  
Vol 12 ◽  
Author(s):  
Rachele Rossi ◽  
Maria Sofia Falzarano ◽  
Hana Osman ◽  
Annarita Armaroli ◽  
Chiara Scotton ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Circadian Rhythm ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Expression Profiles ◽  
Gene Mutations ◽  
Dystrophin Gene ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Muscle Biopsies

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.

scholarly journals The Effect of Deflazacort Treatment on the Functioning of Skeletal Muscle Mitochondria in Duchenne Muscular Dystrophy

2020 ◽  
Vol 21 (22) ◽  
pp. 8763
Author(s):  
Mikhail V. Dubinin ◽  
Eugeny Yu. Talanov ◽  
Kirill S. Tenkov ◽  
Vlada S. Starinets ◽  
Natalia V. Belosludtseva ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Hereditary Disease ◽  
Mdx Mice ◽  
Muscle Mitochondria ◽  
Calcium Uniporter ◽  
Skeletal Muscle Mitochondria ◽  
Pore Opening ◽  
Muscular Function

Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a lack of dystrophin, a protein essential for myocyte integrity. Mitochondrial dysfunction is reportedly responsible for DMD. This study examines the effect of glucocorticoid deflazacort on the functioning of the skeletal-muscle mitochondria of dystrophin-deficient mdx mice and WT animals. Deflazacort administration was found to improve mitochondrial respiration of mdx mice due to an increase in the level of ETC complexes (complexes III and IV and ATP synthase), which may contribute to the normalization of ATP levels in the skeletal muscle of mdx animals. Deflazacort treatment improved the rate of Ca2+ uniport in the skeletal muscle mitochondria of mdx mice, presumably by affecting the subunit composition of the calcium uniporter of organelles. At the same time, deflazacort was found to reduce the resistance of skeletal mitochondria to MPT pore opening, which may be associated with a change in the level of ANT2 and CypD. In this case, deflazacort also affected the mitochondria of WT mice. The paper discusses the mechanisms underlying the effect of deflazacort on the functioning of mitochondria and contributing to the improvement of the muscular function of mdx mice.

scholarly journals Recordings of action potentials, charge movements, and sarcoplasmic reticulum Ca2+ release in isolated adult zebrafish fast skeletal muscle fibers reveals very fast kinetics of excitation–contraction coupling

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Romane Idoux ◽  
Christine Berthier ◽  
Vincent Jacquemond ◽  
Bruno Allard
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Action Potentials ◽  
Muscle Fibers ◽  
Fast Skeletal Muscle ◽  
Fast Kinetics ◽  
Ec Coupling ◽  
Skeletal Muscle Fibers ◽  
Charge Movements ◽  
Kinetics Of

The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation–contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.

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