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1.
There is substantial evidence indicating that disruption of Ca2+ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca2+ deregulation and the Ca2+ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca2+ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca2+ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca2+ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca2+ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca2+ store contributes to the disrupted Ca2+ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy. 相似文献
2.
François-Xavier Boittin George Shapovalov Urs T. Ruegg 《Biochemical and biophysical research communications》2010,391(1):401-406
Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca2+ entry through sarcolemmal cationic channels. Using the Mn2+ quench method, we show here that Mn2+ entry triggered by Ca2+ store depletion but not basal Mn2+ entry relies on Ca2+-independent PLA2 (iPLA2) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx5cv mouse. iPLA2 was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA2 lipid product lysophosphatidylcholine was found to trigger Ca2+ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA2 and lysophospholipid production may be of interest to reduce Ca2+ entry and subsequent degeneration of dystrophic muscle. 相似文献
3.
Cecilia Riquelme María José Acu?a Javiera Torrejón Daniela Rebolledo Daniel Cabrera Robson A. Santos Enrique Brandan 《PloS one》2014,9(4)
Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype. 相似文献
4.
Francisco Altamirano Denisse Valladares Carlos Henríquez-Olguín Mariana Casas Jose R. López Paul D. Allen Enrique Jaimovich 《PloS one》2013,8(12)
Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca2+]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca2+]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca2+]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91phox/p47phox NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca2+]r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD. 相似文献
5.
Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca2+/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg2+-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD+ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD+ in the case of NAD+-linked substrates. 相似文献
6.
《生物化学与生物物理学报:疾病的分子基础》2020,1866(5):165674
Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca2+ homeostasis. This work examines the kinetic parameters of Ca2+ transport and the opening of the Ca2+-dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca2+ uniport, with the kinetics of Na+-dependent Ca2+ efflux not changing. The data obtained indicate that the decreased rate of Ca2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca2+ homeostasis of skeletal-muscle mitochondria. 相似文献
7.
Aline Barbosa Macedo Luis Henrique Rapucci Moraes Daniela Sayuri Mizobuti Aline Reis Foga?a Fernanda dos Santos Rapucci Moraes Tulio de Almeida Hermes Adriana Pertille Elaine Minatel 《PloS one》2015,10(6)
The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i. The mdx group showed significant increase in H2O2 production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies. 相似文献
8.
《BBA》2020,1861(10):148250
Duchenne muscular dystrophy (DMD) is a progressive skeletal muscle disease that is associated with severe cardiac complications in the late stages. Significant mitochondrial dysfunction is reportedly responsible for the development of cardiomyopathy with age. At the same time, adaptive changes in mitochondrial metabolism in cardiomyocytes were identified in the early stages of DMD. In this work, we evaluate the functioning of calcium transport systems (MCU and NCLX), and MPT pore in the heart mitochondria of young dystrophin-deficient mice. As compared to wild-type animals, heart mitochondria of mdx mice have been found to be more efficient both in respect to Ca2+ uniport and Na+-dependent Ca2+ efflux. The data obtained indicate that the increased rate of Ca2+ uptake by heart mitochondria of mdx mice may be due to an increase in the ratio of MCU and MCUb subunits. In turn, an increase in the rate of Ca2+ efflux from organelles in DMD may be the result of a significant increase in the level of NCLX. Moreover, the heart mitochondria of mdx mice were more resistant to MPT pore opening, which may be due to an increase in the microviscosity of mitochondrial membranes of DMD mice. At the same time, the level of putative MPT pore proteins did not change. The paper discusses the effect of rearrangements of the mitochondrial proteome involved in the transport and accumulation of calcium on the adaptation of this organ to DMD. 相似文献
9.
Ki Ho Park Noah Weisleder Jingsong Zhou Kristyn Gumpper Xinyu Zhou Pu Duann Jianjie Ma Pei-Hui Lin 《Journal of visualized experiments : JoVE》2014,(84)
Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+ handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model). 相似文献
10.
Muscle uses Ca2+ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca2+ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca2+ from their surroundings, a process called mitochondrial Ca2+ uptake. Under physiological conditions, Ca2+ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca2+ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca2+ uptake could shape spatio-temporal patterns of intracellular Ca2+ signaling. Malfunction of mitochondrial Ca2+ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca2+ levels. Besides the sudden elevation of Ca2+ level induced by action potentials, Ca2+ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca2+ uptake is fast and big enough to shape intracellular Ca2+ signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca2+ inside mitochondria. This review focuses on characterization of mitochondrial Ca2+ uptake in skeletal muscle and its role in muscle physiology and diseases. 相似文献
11.
The Voltage-dependent Anion Channel in Endoplasmic/Sarcoplasmic Reticulum: Characterization,Modulation and Possible Function 总被引:5,自引:0,他引:5
In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca2+ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca2+and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca2+ homeostasis and mitochondria-mediated apoptosis will be discussed. 相似文献
12.
Alfredo Mijares Francisco Altamirano Juan Kolster José A. Adams José R. López 《Biochemical and biophysical research communications》2014
Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca2+ concentration ([Ca2+]d) and diastolic Na+ concentration ([Na+]d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd3+)-sensitive Ca2+ entry and inositol triphosphate (IP3) signaling pathways in abnormal [Ca2+]d and [Na+]d were investigated. Our results showed an age-dependent increase in both [Ca2+]d and [Na+]d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd3+ treatment significantly reduced both [Ca2+]d and [Na+]d at all ages. In addition, blockade of the IP3-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd3+ normalized both [Ca2+]d and [Na+]d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca2+ and Na+ overload mediated at least in part by enhanced Ca2+ entry through Gd3+ sensitive transient receptor potential channels (TRPC), and by IP3 receptors. 相似文献
13.
Francisco Altamirano Claudio F. Perez Min Liu Jeffrey Widrick Elisabeth R. Barton Paul D. Allen Jose A. Adams Jose R. Lopez 《PloS one》2014,9(9)
Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca2+ and Na+ overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca2+ and Na+ overload, diminished abnormal sarcolemmal Ca2+ entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway. 相似文献
14.
Molecular cascades responsible for Ca2+ homeostasis and Ca2+ signalling could be assembled in highly plastic toolkits that define physiological adaptation of cells to the environment and which are intimately involved in all types of cellular pathology. Control over Ca2+ concentration in different cellular compartments is intimately linked to cell metabolism, because (i) ATP production requires low Ca2+, (ii) Ca2+ homeostatic systems consume ATP and (iii) Ca2+ signals in mitochondria stimulate ATP synthesis being an essential part of excitation–metabolic coupling. The communication between the ER and mitochondria plays an important role in this metabolic fine tuning. In the insulin resistance state and diabetes this communication has been impaired leading to different disorders, for instance, diminished insulin production by pancreatic β cells, reduced heart and skeletal muscle contractility, reduced NO production by endothelial cells, increased glucose production by liver, increased lipolysis by adipose cells, reduced immune responses, reduced cognitive functions, among others. All these processes eventually trigger degenerative events resulting in overt diabetes due to reduction of pancreatic β cell mass, and different complications of diabetes, such as retinopathy, nephropathy, neuropathy, and different cardiovascular diseases. 相似文献
15.
In skeletal muscle, dysfunctional contractile activity has been linked to impaired intracellular Ca2+ concentration ([Ca2+]i) regulation. Muscle force production is impaired and fatigability and muscle fragility deteriorate with diabetes. Use of a novel in vivo model permits investigation of [Ca2+]i homeostasis in diabetic skeletal muscle. Within this in vivo environment we have shown that diabetes perturbs the Ca2+ regulatory system such that resting [Ca2+]i homeostasis following muscle contractions is compromised and elevations of [Ca2+]i are exacerbated. This review considers the impact of diabetes on the capacity of skeletal muscle to regulate [Ca2+]i, following muscle contractions and, in particular, the relationship between muscle fatigue and elevated [Ca2+]i in a highly ecologically relevant circulation-intact environment. Importantly, the role of mitochondria in calcium sequestration and the possibility that diabetes impacts this process is explored. Given the profound microcirculatory dysfunction in diabetes this preparation offers the unique opportunity to study the interrelationships among microvascular function, blood-myocyte oxygen flux and [Ca2+]i as they relate to enhanced muscle fatigability and exercise intolerance. 相似文献
16.
The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B1 (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca2+ efflux and swelling in mitochondria in the absence of phosphate, Mg2+ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca2+ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phopsphate and Mg2+ added together, and partially inhibited with phosphate plus either ATP or Mg2+. Between 0 and 7 μM added Ca2+ and in the presence of phosphate, ATP and Mg2+, tri-Calciphor stimulated the uptake of Ca2+ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca2+. Thus depending on the assay conditions, two different effects involving Ca2+ movements and mitochondria are observed with tri-Calciphor. 相似文献
17.
Glen B. Banks Ariana C. Combs Guy L. Odom Robert J. Bloch Jeffrey S. Chamberlain 《PLoS genetics》2014,10(6)
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin−/− mice. An ∼2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin−/−, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD. 相似文献
18.
Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. Nol3 (Arc protein) genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3
-/-
Sgcd
-/- double targeted mice. Mechanistically, Nol3
-/-
Sgcd
-/- mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms. 相似文献
19.
Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches. 相似文献
20.
Calcium uptake of cardiac muscle and fast-twitch and slow-twitch skeletal muscle of rabbit was measured in 10-μm thin sections. Ca2+ uptake showed K+, Mg2+, Ca2+, and oxalate dependence and required ATP. The contribution of mitochondria to the Ca2+ uptake could be ruled out, since inclusion of ruthenium red or sodium azide in the medium did not show inhibition. The method, which avoids unphysiological fragmentation of the sarcoplasmic reticulum, has the added advantage of requiring only 0.1 to 0.3 g of muscle and permitting simultaneous histochemical studies from the same muscle block. 相似文献