RESUMO
Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout (Krt18-KO) or dominant-negative mutant mice (Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.
Assuntos
Filamentos Intermediários/metabolismo , Contração Isométrica , Queratina-18/metabolismo , Força Muscular , Músculo Esquelético/metabolismo , Animais , Feminino , Filamentos Intermediários/ultraestrutura , Queratina-18/deficiência , Queratina-18/genética , Queratina-19/genética , Queratina-19/metabolismo , Masculino , Camundongos Knockout , Músculo Esquelético/ultraestrutura , Transdução de SinaisRESUMO
Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1(null) IL2rγ(null) immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics.
Assuntos
Marcadores Genéticos , Xenoenxertos/fisiologia , Músculo Esquelético/transplante , Distrofia Muscular Facioescapuloumeral/cirurgia , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/patologiaRESUMO
Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated significant alterations in the neuromuscular junction (NMJ) morphology and resulting neuromuscular transmission failure (NTF) 24 h after injury in mdx mice (murine model for DMD). Here we determine the contribution of NMJ morphology and NTF to the recovery of muscle contractile function post-injury. NMJ morphology and NTF rates were assessed day 0 (immediately after injury) and days 1, 7, 14 and 21 after quadriceps injury. Eccentric injury of the quadriceps resulted in a significant loss of maximal torque in both WT (39 ± 6 %) and mdx (76 ± 8 %) with a full recovery in WT by day 7 and in mdx by day 21. Post-injury alterations in NMJ morphology and NTF were found only in mdx, were limited to days 0 and 1, and were independent of changes in MuSK or AChR expression. Such early changes at the NMJ after injury are consistent with mechanical disruption rather than newly forming NMJs. Furthermore, we show that the dense microtubule network that underlies the NMJ is significantly reduced and disorganized in mdx compared to WT. These structural changes at the NMJ may play a role in the increased NMJ disruption and the exaggerated loss of nerve-evoked muscle force seen after injury to dystrophic muscles.
Assuntos
Distrofina/fisiologia , Distrofia Muscular de Duchenne/patologia , Junção Neuromuscular/lesões , Junção Neuromuscular/metabolismo , Regeneração/fisiologia , Animais , Western Blotting , Células Cultivadas , Imunofluorescência , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Junção Neuromuscular/fisiopatologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Colinérgicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin's association with calcium (Ca(2+)) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca(2+)] or blocking L-type Ca(2+) channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Estresse Fisiológico , Animais , Anti-Hipertensivos/farmacologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Membrana Celular/patologia , Diltiazem/farmacologia , Disferlina , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Contração Muscular/efeitos dos fármacos , Contração Muscular/genética , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Necrose/genética , Necrose/metabolismo , Necrose/patologiaRESUMO
Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α-actinin and vinculin. Here we examine the roles and expression of the α and ß forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell-cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α-synemin preferentially localized to cell-cell junctions, whereas ß-synemin was primarily at the level of Z disks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell-cell junctions, with no effect on sarcomeric organization. Solubility assays showed that ß-synemin was soluble and interacted with sarcomeric α-actinin by coimmunoprecipitation, while α-synemin and desmin were insoluble. We conclude that ß-synemin mediates the association of desmin IFs with Z disks, whereas α-synemin stabilizes junctional complexes between cardiomyocytes.
Assuntos
Desmina/fisiologia , Junções Intercelulares/fisiologia , Proteínas de Filamentos Intermediários/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Actinina/metabolismo , Animais , Animais Recém-Nascidos , Adesão Celular/fisiologia , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/genética , Filamentos Intermediários/fisiologia , Isomerismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Sarcômeros/fisiologia , Solubilidade , Vinculina/metabolismoRESUMO
During aging, the regenerative capacity of muscle stem cells (MuSCs) decreases, diminishing the ability of muscle to repair following injury. We found that the ability of MuSCs to regenerate is regulated by the primary cilium, a cellular protrusion that serves as a sensitive sensory organelle. Abolishing MuSC cilia inhibited MuSC proliferation in vitro and severely impaired injury-induced muscle regeneration in vivo. In aged muscle, a cell intrinsic defect in MuSC ciliation was associated with the decrease in regenerative capacity. Exogenous activation of Hedgehog signaling, known to be localized in the primary cilium, promoted MuSC expansion, both in vitro and in vivo. Delivery of the small molecule Smoothened agonist (SAG1.3) to muscles of aged mice restored regenerative capacity leading to increased strength post-injury. These findings provide fresh insights into the signaling dysfunction in aged MuSCs and identify the ciliary Hedgehog signaling pathway as a potential therapeutic target to counter the loss of muscle regenerative capacity which accompanies aging.
Assuntos
Cílios , Músculo Esquelético , Envelhecimento/fisiologia , Animais , Proteínas Hedgehog , Camundongos , Músculo Esquelético/fisiologia , MioblastosRESUMO
Stromal interaction molecule 1 (STIM1), the sarcoplasmic reticulum (SR) transmembrane protein, activates store-operated Ca2+ entry (SOCE) in skeletal muscle and, thereby, coordinates Ca2+ homeostasis, Ca2+-dependent gene expression, and contractility. STIM1 occupies space in the junctional SR membrane of the triads and the longitudinal SR at the Z-line. How STIM1 is organized and is retained in these specific subdomains of the SR is unclear. Here, we identified desmin, the major type III intermediate filament protein in muscle, as a binding partner for STIM1 based on a yeast 2-hybrid screen. Validation of the desmin-STIM1 interaction by immunoprecipitation and immunolocalization confirmed that the CC1-SOAR domains of STIM1 interact with desmin to enhance STIM1 oligomerization yet limit SOCE. Based on our studies of desmin-KO mice, we developed a model wherein desmin connected STIM1 at the Z-line in order to regulate the efficiency of Ca2+ refilling of the SR. Taken together, these studies showed that desmin-STIM1 assembles a cytoskeletal-SR connection that is important for Ca2+ signaling in skeletal muscle.
Assuntos
Desmina/genética , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , RNA/genética , Molécula 1 de Interação Estromal/genética , Animais , Sinalização do Cálcio , Células Cultivadas , Desmina/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Animais , Músculo Esquelético/ultraestrutura , Retículo Sarcoplasmático/metabolismo , Molécula 1 de Interação Estromal/biossínteseRESUMO
This corrects the article DOI: 10.1038/srep12637.
RESUMO
Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.
Assuntos
Técnicas Biossensoriais , Polarização de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cor , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Citosol/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/instrumentação , Corantes Fluorescentes/síntese química , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Transfecção , Quinases da Família src/genética , Quinases da Família src/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: In muscular dystrophy and old age, skeletal muscle repair is compromised leading to fibrosis and fatty tissue accumulation. Therefore, therapies that protect skeletal muscle or enhance repair would be valuable medical treatments. Hypoxia-inducible factors (HIFs) regulate gene transcription under conditions of low oxygen, and HIF target genes EPO and VEGF have been associated with muscle protection and repair. We tested the importance of HIF activation following skeletal muscle injury, in both a murine model and human volunteers, using prolyl hydroxylase inhibitors that stabilize and activate HIF. METHODS: Using a mouse eccentric limb injury model, we characterized the protective effects of prolyl hydroxylase inhibitor, GSK1120360A. We then extended these studies to examine the impact of EPO modulation and infiltrating immune cell populations on muscle protection. Finally, we extended this study with an experimental medicine approach using eccentric arm exercise in untrained volunteers to measure the muscle-protective effects of a clinical prolyl hydroxylase inhibitor, daprodustat. RESULTS: GSK1120360A dramatically prevented functional deficits and histological damage, while accelerating recovery after eccentric limb injury in mice. Surprisingly, this effect was independent of EPO, but required myeloid HIF1α-mediated iNOS activity. Treatment of healthy human volunteers with high-dose daprodustat reduced accumulation of circulating damage markers following eccentric arm exercise, although we did not observe any diminution of functional deficits with compound treatment. CONCLUSION: The results of these experiments highlight a novel skeletal muscle protective effect of prolyl hydroxylase inhibition via HIF-mediated expression of iNOS in macrophages. Partial recapitulation of these findings in healthy volunteers suggests elements of consistent pharmacology compared to responses in mice although there are clear differences between these two systems.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Glicina/análogos & derivados , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Contração Muscular , Músculo Esquelético/efeitos dos fármacos , Mialgia/tratamento farmacológico , Quinolonas/uso terapêutico , Adolescente , Adulto , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Glicina/farmacologia , Glicina/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Mialgia/etiologia , Quinolonas/farmacologiaRESUMO
Following publication of the original article [1], the authors flagged that there is a discrepancy with the Availability of data and materials statement on page 12 of the article.
RESUMO
The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely by regulating the abundance of sclerostin, a secreted inhibitor of bone formation. We defined a microtubule-dependent mechanotransduction pathway that linked fluid shear stress to reactive oxygen species (ROS) and calcium (Ca2+) signals that led to a reduction in sclerostin abundance in cultured osteocytes. We demonstrated that microtubules stabilized by detyrosination, a reversible posttranslational modification of polymerized α-tubulin, determined the stiffness of the cytoskeleton, which set the mechanoresponsive range of cultured osteocytes to fluid shear stress. We showed that fluid shear stress through the microtubule network activated NADPH oxidase 2 (NOX2)-generated ROS that target the Ca2+ channel TRPV4 to elicit Ca2+ influx. Furthermore, tuning the abundance of detyrosinated tubulin affected cytoskeletal stiffness to define the mechanoresponsive range of cultured osteocytes to fluid shear stress. Finally, we demonstrated that NOX2-ROS elicited Ca2+ signals that activated the kinase CaMKII to decrease the abundance of sclerostin protein. Together, these discoveries may identify potentially druggable targets for regulating osteocyte mechanotransduction to affect bone quality.
Assuntos
Glicoproteínas/metabolismo , Mecanotransdução Celular , Microtúbulos/fisiologia , NADPH Oxidase 2/metabolismo , Osteócitos/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Microtúbulos/química , Microtúbulos/ultraestrutura , NADPH Oxidase 2/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPV/fisiologia , Tubulina (Proteína)/análiseRESUMO
We report here a method for the use of poly-l-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research.
RESUMO
Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cálcio/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Proteoma/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
Myosin Binding Protein-C slow (sMyBP-C) is expressed in skeletal muscles where it plays structural and regulatory roles. The functions of sMyBP-C are modulated through alternative splicing and phosphorylation. Herein, we examined the phosphorylation profile of sMyBP-C in mouse slow-twitch soleus muscle isolated from fatigued or non-fatigued young (2-4-months old) and old (~14-months old) wild type and mdx mice. Our findings are two-fold. First, we identified the phosphorylation events present in individual sMyBP-C variants at different states. Secondly, we quantified the relative abundance of each phosphorylation event, and of sMyBP-C phospho-species as a function of age and dystrophy, in the presence or absence of fatigue. Our results revealed both constitutive and differential phosphorylation of sMyBP-C. Moreover, we noted a 10-40% and a 25-35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx soleus muscles, respectively. On the contrary, we observed a 5-10% and a 20-25% increase in the phosphorylation levels of specific sites in young fatigued wild type and mdx soleus muscles, respectively. Overall, our studies showed that the phosphorylation pattern of sMyBP-C is differentially regulated following reversible (i.e. fatigue) and non-reversible (i.e. age and disease) (patho)physiological stressors.
Assuntos
Proteínas de Transporte/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Doenças Musculares/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/química , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fadiga Muscular , Doenças Musculares/patologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , FosforilaçãoRESUMO
In striated muscle, X-ROS is the mechanotransduction pathway by which mechanical stress transduced by the microtubule network elicits reactive oxygen species. X-ROS tunes Ca(2+) signalling in healthy muscle, but in diseases such as Duchenne muscular dystrophy (DMD), microtubule alterations drive elevated X-ROS, disrupting Ca(2+) homeostasis and impairing function. Here we show that detyrosination, a post-translational modification of α-tubulin, influences X-ROS signalling, contraction speed and cytoskeletal mechanics. In the mdx mouse model of DMD, the pharmacological reduction of detyrosination in vitro ablates aberrant X-ROS and Ca(2+) signalling, and in vivo it protects against hallmarks of DMD, including workload-induced arrhythmias and contraction-induced injury in skeletal muscle. We conclude that detyrosinated microtubules increase cytoskeletal stiffness and mechanotransduction in striated muscle and that targeting this post-translational modification may have broad therapeutic potential in muscular dystrophies.
Assuntos
Microtúbulos/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Fenômenos Biomecânicos , Cálcio , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Microscopia de Força Atômica , Ratos , Ratos Sprague-DawleyRESUMO
The class of muscular dystrophies linked to the genetic ablation or mutation of dysferlin, including Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy (MM), are late-onset degenerative diseases. In lieu of a genetic cure, treatments to prevent or slow the progression of dysferlinopathy are of the utmost importance. Recent advances in the study of dysferlinopathy have highlighted the necessity for the maintenance of calcium handling in altering or slowing the progression of muscular degeneration resulting from the loss of dysferlin. This review highlights new evidence for a role for dysferlin at the transverse (t-) tubule of striated muscle, where it is involved in maintaining t-tubule structure and function.
RESUMO
Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently, we discovered a pathway by which mechanical stretch activates NADPH Oxidase 2 (Nox2) dependent ROS generation (X-ROS). Our work in dystrophic skeletal muscle revealed that X-ROS is excessive in dystrophin-deficient (mdx) skeletal muscle and contributes to muscle injury susceptibility, a hallmark of the dystrophic process. We also observed widespread alterations in the expression of genes associated with the X-ROS pathway and redox homeostasis in muscles from both Duchenne muscular dystrophy patients and mdx mice. As nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the transcriptional regulation of genes involved in redox homeostasis, we hypothesized that Nrf2 deficiency may contribute to enhanced X-ROS signaling by reducing redox buffering. To directly test the effect of diminished Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy-a model with a mild histopathologic and functional phenotype. Nrf2-deficient A/J mice exhibited significant muscle-specific functional deficits, histopathologic abnormalities, and dramatically enhanced X-ROS compared to control A/J and WT mice, both with functional Nrf2. Having identified that reduced Nrf2 activity is a negative disease modifier, we propose that strategies targeting Nrf2 activation may address the generalized reduction in redox homeostasis to halt or slow dystrophic progression.