scholarly journals TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Anna Creisméas ◽  
Claire Gazaille ◽  
Audrey Bourdon ◽  
Marc-Antoine Lallemand ◽  
Virginie François ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Calcium Homeostasis ◽  
Intracellular Localization ◽  
Muscle Fibers ◽  
Specific Inhibitor ◽  
Apparent Molecular Weight ◽  
Complementary Therapy ◽  
Cytosolic Calcium ◽  
Inherited Disease ◽  
Protein Levels

Abstract Background Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD. Methods All experiments were conducted in the DMDmdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca2+]c) and SPCa in EDL (Extensor Digitorum Longus) muscle fibers from age-matched WT and DMDmdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis. Results As expected from the malignant hyperthermia like episodes observed in several DMDmdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca2+]c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMDmdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMDmdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMDmdx. Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase. Conclusions All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.

scholarly journals Distribution and activation of matrix metalloproteinase-2 in skeletal muscle fibers

2019 ◽  
Vol 317 (3) ◽  
pp. C613-C625 ◽  
Author(s):  
Xiaoyu Ren ◽  
Graham D. Lamb ◽  
Robyn M. Murphy
Keyword(s):  
Skeletal Muscle ◽  
Matrix Metalloproteinase ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Intracellular Localization ◽  
Muscle Fibers ◽  
Matrix Metalloproteinase 2 ◽  
Absolute Amount ◽  
Skinned Muscle ◽  
Functional Relevance

A substantial intracellular localization of matrix metalloproteinase 2 (MMP2) has been reported in cardiomyocytes, where it plays a role in the degradation of the contractile apparatus following ischemia-reperfusion injury. Whether MMP2 may have a similar function in skeletal muscle is unknown. This study determined that the absolute amount of MMP2 is similar in rat skeletal and cardiac muscle and human muscle (~10–18 nmol/kg muscle wet wt) but is ~50- to 100-fold less than the amount of calpain-1. We compared mechanically skinned muscle fibers, where the extracellular matrix (ECM) is completely removed, with intact fiber segments and found that ~30% of total MMP2 was associated with the ECM, whereas ~70% was inside the muscle fibers. Concordant with whole muscle fractionation, further separation of skinned fiber segments into cytosolic, membranous, and cytoskeletal and nuclear compartments indicated that ~57% of the intracellular MMP2 was freely diffusible, ~6% was associated with the membrane, and ~37% was bound within the fiber. Under native zymography conditions, only 10% of MMP2 became active upon prolonged (17 h) exposure to 20 μM Ca2+, a concentration that would fully activate calpain-1 in seconds to minutes; full activation of MMP2 would require ~1 mM Ca2+. Given the prevalence of intracellular MMP2 in skeletal muscle, it is necessary to investigate its function using physiological conditions, including isolation of any potential functional relevance of MMP2 from that of the abundant protease calpain-1.

Chimeric calsequestrin and its targeting to the junctional sarcoplasmic reticulum of skeletal muscle

1997 ◽  
Vol 272 (5) ◽  
pp. C1420-C1428 ◽  
Author(s):  
A. Nori ◽  
K. A. Nadalini ◽  
A. Martini ◽  
R. Rizzuto ◽  
A. Villa ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Soleus Muscle ◽  
Hela Cells ◽  
Skeletal Muscles ◽  
Intracellular Localization ◽  
Primary Cultures ◽  
Muscle Fibers ◽  
Junctional Sarcoplasmic Reticulum

Calsequestrin (CS) is the junctional sarcoplasmic reticulum (jSR) Ca2+ binding protein responsible for intraluminal Ca2+ storage. The targeting mechanisms of CS to the jSR are yet to be unraveled. The nine-amino acid epitope of the influenza virus hemoagglutinin (referred to as HA1) was added at the COOH-terminal of CS by polymerase chain reaction cloning. The HA1-tagged CS cDNA was transiently transfected in either HeLa cells, myogenic cell lines, such as C2 and L8 cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of chimeric CS-HA1 were monitored by epifluorescence and confocal microscopy using either anti-CS antibodies or anti-HA1 antibodies. About 30% of transfected HeLa cells and 20-40% of myogenic cells expressed CS-HA1 into intracellular compartments, such as the perinuclear cisternae of endoplasmic reticulum (ER). Myoblasts of newborn rat skeletal muscles were first transfected and subsequently stimulated to differentiate into myotubes. CS-HA1 was detected in approximately 20% of transfected myotubes and did not affect CS distribution in myotubes. In the soleus muscle of adult rat, intramuscular injection of bupivacaine induced necrosis followed by regeneration. In vivo transfection of HA1-tagged CS cDNA in regenerating skeletal muscles determined expression in a few skeletal muscle fibers; CS-HA1 was localized only in jSR, as judged by confocal microscopy of longitudinal sections. The present results show that chimeric CS-HA1 is correctly sorted to ER/SR compartments and that the free COOH-terminal is not requested for sorting, retention, and segregation of CS to the SR.

Expression of the Na+/Ca2+exchanger in skeletal muscle

2001 ◽  
Vol 280 (1) ◽  
pp. C146-C154 ◽  
Author(s):  
Bodvaël Fraysse ◽  
Thierry Rouaud ◽  
Marie Millour ◽  
Josiane Fontaine-Pérus ◽  
Marie-France Gardahaut ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Cellular Level ◽  
Postnatal Life ◽  
Fiber Types ◽  
Developmentally Regulated ◽  
Protein Levels ◽  
Isoform Expression ◽  
Fast Twitch

The expression of the Na+/Ca2+ exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na+/Ca2+ exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.

Rabbits fed cholesterol-enriched diets exhibit pathological features of inclusion body myositis

2008 ◽  
Vol 294 (3) ◽  
pp. R829-R835 ◽  
Author(s):  
Xuesong Chen ◽  
Othman Ghribi ◽  
Jonathan D. Geiger
Keyword(s):  
Alzheimer’S Disease ◽  
Skeletal Muscle ◽  
Alzheimer's Disease ◽  
Inclusion Body ◽  
Inclusion Body Myositis ◽  
Muscle Fibers ◽  
Muscle Disease ◽  
Amyloid Β Protein ◽  
Pathological Features ◽  
Protein Levels

Sporadic inclusion body myositis (IBM) is the most common age-related muscle disease in humans; however, its etiology is unknown, there are few animal models for this disease, and effective treatments have not been identified. Similarities between pathological findings in Alzheimer's disease brain and IBM skeletal muscle include increased levels of amyloid precursor protein (APP) and amyloid β-protein (Aβ). Moreover, there have been suggestions that elevated levels of free cholesterol might participate in the pathogenesis of Alzheimer's disease and IBM due, in part, to its role in Aβ generation. Here, we tested the hypothesis that rabbits fed cholesterol-enriched diets might faithfully exhibit human-like IBM pathological features. In skeletal muscle of one-third of the female rabbits fed cholesterol-enriched diet but not control diet, we found features of IBM, including vacuolated muscle fibers, increased numbers of mononuclear inflammatory cells, increased intramuscular deposition of Aβ, hyperphosphorylated tau, and increased numbers of muscle fibers immunopositive for ubiquitin. The cholesterol-enriched diet increased mRNA and protein levels of APP, increased the protein levels of βAPP cleaving enzyme, and shifted APP processing in favor of Aβ production. Our study has demonstrated that increased ingestion of high levels of dietary cholesterol can result in pathological features that resemble IBM closely and thus may serve as an important new model with which to study this debilitating disorder.

scholarly journals Disturbances in Calcium Homeostasis Promotes Skeletal Muscle Atrophy: Lessons From Ventilator-Induced Diaphragm Wasting

2020 ◽  
Vol 11 ◽  
Author(s):  
Hayden W. Hyatt ◽  
Scott K. Powers
Keyword(s):  
Calcium Homeostasis ◽  
Calcium Release ◽  
Muscle Fibers ◽  
Clinical Problem ◽  
Cytosolic Calcium ◽  
Diaphragm Muscle ◽  
Skeletal Muscle Atrophy ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Diaphragmatic Weakness

Mechanical ventilation (MV) is often a life-saving intervention for patients in respiratory failure. Unfortunately, a common and undesired consequence of prolonged MV is the development of diaphragmatic atrophy and contractile dysfunction. This MV-induced diaphragmatic weakness is commonly labeled “ventilator-induced diaphragm dysfunction” (VIDD). VIDD is an important clinical problem because diaphragmatic weakness is a major risk factor for the failure to wean patients from MV; this inability to remove patients from ventilator support results in prolonged hospitalization and increased morbidity and mortality. Although several processes contribute to the development of VIDD, it is clear that oxidative stress leading to the rapid activation of proteases is a primary contributor. While all major proteolytic systems likely contribute to VIDD, emerging evidence reveals that activation of the calcium-activated protease calpain plays a required role. This review highlights the signaling pathways leading to VIDD with a focus on the cellular events that promote increased cytosolic calcium levels and the subsequent activation of calpain within diaphragm muscle fibers. In particular, we discuss the emerging evidence that increased mitochondrial production of reactive oxygen species promotes oxidation of the ryanodine receptor/calcium release channel, resulting in calcium release from the sarcoplasmic reticulum, accelerated proteolysis, and VIDD. We conclude with a discussion of important and unanswered questions associated with disturbances in calcium homeostasis in diaphragm muscle fibers during prolonged MV.

scholarly journals Cytosolic calcium transients are a determinant of contraction-induced HSP72 transcription in single skeletal muscle fibers

2016 ◽  
Vol 120 (10) ◽  
pp. 1260-1266 ◽  
Author(s):  
Creed M. Stary ◽  
Michael C. Hogan
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Cytosolic Calcium ◽  
Mrna Levels ◽  
Amp Kinase ◽  
Related Factors ◽  
Real Time Quantitative Pcr ◽  
Cross Bridge ◽  
Skeletal Muscle Fibers ◽  
Fatigue Development

The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca2+ transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca2+ transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca2+ concentration ([Ca2+]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca2+]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl- p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca2+]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: <3 mmHg) or AMP kinase activation had no effect on HSP72 mRNA levels. These results suggest that the intermittent cytosolic Ca2+ transient that occurs with skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role.

scholarly journals Numb is required for optimal contraction of skeletal muscle

2021 ◽  
Author(s):  
Rita De Gasperi ◽  
Chenglin Mo ◽  
Daniella Azulai ◽  
Zhiying Wang ◽  
Lauren M Harlow ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Fate ◽  
Cell Fusion ◽  
Mitochondrial Function ◽  
Calcium Release ◽  
Ion Beam ◽  
Muscle Fibers ◽  
Cytosolic Calcium ◽  
Adult Skeletal Muscle ◽  
Primary Mouse

Background: The role of Numb, a protein that is important for cell fate and development was investigated in adult skeletal muscle in mice using a conditional, inducible knockout (cKO) model. Methods: Numb expression was evaluated by Western blot. Numb localization was determined by confocal microscopy. The effects of cKO of Numb and the closely-related gene Numb-like in skeletal muscle fibers was evaluated by in-situ physiology; transmission and focused ion beam scanning electron microscopy; 3-dimensional reconstruction of mitochondrial; lipidomics; and bulk RNA-sequencing. Additional studies using primary mouse myotubes investigated the effects the effects of Numb knockdown on cell fusion, mitochondrial function and calcium transients. Results: Numb protein expression was reduced by ~70% (p < 0.01) at 24 as compared to 3 months of age. Numb was localized within muscle fibers as bands traversing fibers at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb-like reduced specific tetanic force by 36%, p < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (p < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (p < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1 and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acid (PUFAs) through lipoxygenases were upregulated in Numb cKO skeletal muscle; 12-HEPE was increased by ~250% (p < 0.05) and 17,18-EpETE by ~240% (p < 0.05). In mouse primary myotubes, Numb knock-down reduced cell fusion (~20%, p < 0.01) and mitochondrial function and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (p < 0.01). Conclusions: These findings implicate Numb as a critical factor in skeletal muscle structure and function which appear to be critical for calcium release; we therefore speculate Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

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