RESUMO
ΔR4-R23/ΔCT micro-dystrophin (µDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, µDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of µDys, we compared by mass spectrometry the composition of purified dystrophin and µDys protein complexes in the mouse heart. We report that compared to dystrophin, µDys has altered associations with α1- and ß2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of µDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.
Assuntos
Cardiomiopatias , Distrofia Muscular de Duchenne , Animais , Cardiomiopatias/genética , Distrofina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/metabolismo , ProteômicaRESUMO
Syntrophins are a family of modular adaptor proteins that are part of the dystrophin protein complex, where they recruit and anchor a variety of signaling proteins. Previously we generated mice lacking α- and/or ß2-syntrophin but showed that in the absence of one isoform, other syntrophin isoforms can partially compensate. Therefore, in the current study, we generated mice that lacked α, ß1 and ß2-syntrophins [triple syntrophin knockout (tKO) mice] and assessed skeletal and cardiac muscle function. The tKO mice showed a profound reduction in voluntary wheel running activity at both 6 and 12 months of age. Function of the tibialis anterior was assessed in situ and we found that the specific force of tKO muscle was decreased by 20-25% compared with wild-type mice. This decrease was accompanied by a shift in fiber-type composition from fast 2B to more oxidative fast 2A fibers. Using echocardiography to measure cardiac function, it was revealed that tKO hearts had left ventricular cardiac dysfunction and were hypertrophic, with a thicker left ventricular posterior wall. Interestingly, we also found that membrane-localized dystrophin expression was lower in both skeletal and cardiac muscles of tKO mice. Since dystrophin mRNA levels were not different in tKO, this finding suggests that syntrophins may regulate dystrophin trafficking to, or stabilization at, the sarcolemma. These results show that the loss of all three major muscle syntrophins has a profound effect on exercise performance, and skeletal and cardiac muscle dysfunction contributes to this deficiency.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Proteínas Associadas à Distrofina/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Distrofina/genética , Distrofina/fisiologia , Proteínas Associadas à Distrofina/genética , Coração/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Miocárdio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologiaRESUMO
Mutation of the gene encoding dystrophin leads to Duchenne and Becker muscular dystrophy (DMD and BMD). Currently, dystrophin is thought to function primarily as a structural protein, connecting the muscle cell actin cytoskeleton to the extra-cellular matrix. In addition to this structural role, dystrophin also plays an important role as a scaffold that organizes an array of signaling proteins including sodium, potassium, and calcium channels, kinases, and nitric oxide synthase (nNOS). Many of these signaling proteins are linked to dystrophin via syntrophin, an adapter protein that is known to bind directly to two sites in the carboxyl terminal region of dystrophin. A search of the dystrophin sequence revealed three additional potential syntrophin binding sites (SBSs) within the spectrin-like repeat (SLR) region of dystrophin. Binding assays revealed that the site at SLR 17 bound specifically to the α isoform of syntrophin while the site at SLR 22 bound specifically to the ß-syntrophins. The SLR 17 α-SBS contained the core sequence known to be required for nNOS-dystrophin interaction. In vitro and in vivo assays indicate that α-syntrophin facilitates the nNOS-dystrophin interaction at this site rather than nNOS binding directly to dystrophin as previously reported. The identification of multiple SBSs within the SLR region of dystrophin demonstrates that this region functions as a signaling scaffold. The signaling role of the SLR region of dystrophin will need to be considered for effective gene replacement or exon skipping based DMD/BMD therapies.
Assuntos
Proteínas Associadas à Distrofina/metabolismo , Distrofina/metabolismo , Óxido Nítrico Sintase Tipo I/fisiologia , Sequências Repetitivas de Aminoácidos , Espectrina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Associadas à Distrofina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Homologia de Sequência , Espectrina/químicaRESUMO
Proper function of the retina depends heavily on a specialized form of retinal glia called Müller cells. These cells carry out important homeostatic functions that are contingent on their polarized nature. Specifically, the Müller cell endfeet that contact retinal microvessels and the corpus vitreum show a tenfold higher concentration of the inwardly rectifying potassium channel Kir 4.1 than other Müller cell plasma membrane domains. This highly selective enrichment of Kir 4.1 allows K+ to be siphoned through endfoot membranes in a special form of spatial buffering. Here, we show that Kir 4.1 is enriched in endfoot membranes through an interaction with ß1-syntrophin. Targeted disruption of this syntrophin caused a loss of Kir 4.1 from Müller cell endfeet without affecting the total level of Kir 4.1 expression in the retina. Targeted disruption of α1-syntrophin had no effect on Kir 4.1 localization. Our findings show that the Kir 4.1 aggregation that forms the basis for K+ siphoning depends on a specific syntrophin isoform that colocalizes with Kir 4.1 in Müller endfoot membranes.
Assuntos
Proteínas Associadas à Distrofina/deficiência , Células Ependimogliais/metabolismo , Deleção de Genes , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Retina/metabolismo , Animais , Proteínas Associadas à Distrofina/genética , Células Ependimogliais/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio Corretores do Fluxo de Internalização/genética , Agregados Proteicos/fisiologia , Retina/patologiaRESUMO
Nitric oxide (NO) is a key regulator of skeletal muscle function and metabolism, including vasoregulation, mitochondrial function, glucose uptake, fatigue and excitation-contraction coupling. The main generator of NO in skeletal muscle is the muscle-specific form of neuronal nitric oxide synthase (nNOSµ) produced by the NOS1 gene. Skeletal muscle nNOSµ is predominantly localized at the sarcolemma by interaction with the dystrophin protein complex (DPC). In Duchenne muscular dystrophy (DMD), loss of dystrophin leads to the mislocalization of nNOSµ from the sarcolemma to the cytosol. This perturbation has been shown to impair contractile function and cause muscle fatigue in dystrophic (mdx) mice. Here, we investigated the effect of restoring sarcolemmal nNOSµ on muscle contractile function in mdx mice. To achieve this, we designed a modified form of nNOSµ (NOS-M) that is targeted to the sarcolemma by palmitoylation, even in the absence of the DPC. When expressed specifically in mdx skeletal muscle, NOS-M significantly attenuates force loss owing to damaging eccentric contractions and repetitive isometric contractions (fatigue), while also improving force recovery after fatigue. Expression of unmodified nNOSµ at similar levels does not lead to sarcolemmal association and fails to improve muscle function. Aside from the benefits of sarcolemmal-localized NO production, NOS-M also increased the surface membrane levels of utrophin and other DPC proteins, including ß-dystroglycan, α-syntrophin and α-dystrobrevin in mdx muscle. These results suggest that the expression of NOS-M in skeletal muscle may be therapeutically beneficial in DMD and other muscle diseases characterized by the loss of nNOSµ from the sarcolemma.
Assuntos
Contração Muscular , Óxido Nítrico Sintase Tipo I/metabolismo , Sarcolema/metabolismo , Animais , Proteínas Associadas à Distrofina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Utrofina/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease with no effective treatment. DMD muscle pathogenesis is characterized by chronic inflammation, oxidative stress, and fibrosis. Statins, cholesterol-lowering drugs, inhibit these deleterious processes in ischemic diseases affecting skeletal muscle, and therefore have potential to improve DMD. However, statins have not been considered for DMD, or other muscular dystrophies, principally because skeletal-muscle-related symptoms are rare, but widely publicized, side effects of these drugs. Here we show positive effects of statins in dystrophic skeletal muscle. Simvastatin dramatically reduced damage and enhanced muscle function in dystrophic (mdx) mice. Long-term simvastatin treatment vastly improved overall muscle health in mdx mice, reducing plasma creatine kinase activity, an established measure of muscle damage, to near-normal levels. This reduction was accompanied by reduced inflammation, more oxidative muscle fibers, and improved strength of the weak diaphragm muscle. Shorter-term treatment protected against muscle fatigue and increased mdx hindlimb muscle force by 40%, a value comparable to current dystrophin gene-based therapies. Increased force correlated with reduced NADPH Oxidase 2 protein expression, the major source of oxidative stress in dystrophic muscle. Finally, in old mdx mice with severe muscle degeneration, simvastatin enhanced diaphragm force and halved fibrosis, a major cause of functional decline in DMD. These improvements were accompanied by autophagy activation, a recent therapeutic target for DMD, and less oxidative stress. Together, our findings highlight that simvastatin substantially improves the overall health and function of dystrophic skeletal muscles and may provide an unexpected, novel therapy for DMD and related neuromuscular diseases.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fibras Musculares Esqueléticas , Força Muscular/efeitos dos fármacos , Distrofia Muscular de Duchenne , Sinvastatina/farmacologia , Animais , Creatina Quinase/sangue , Masculino , Camundongos , Camundongos Endogâmicos mdx , Complexos Multienzimáticos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , NADH NADPH Oxirredutases/metabolismo , Oxirredução/efeitos dos fármacosRESUMO
KEY POINTS: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease that is commonly studied using the mdx mouse. The mdx diaphragm muscle closely mimics the pathophysiological changes in DMD muscles. mdx diaphragm force is commonly assessed ex vivo, precluding time course studies. Here we used ultrasonography to evaluate time-dependent changes in diaphragm function in vivo, by measuring diaphragm movement amplitude. In mdx mice, diaphragm amplitude decreased with age and values were much lower than for wild-type mice. Importantly, diaphragm amplitude strongly correlated with ex vivo specific force values. Micro-dystrophin administration increased mdx diaphragm amplitude by 26% after 4 weeks. Diaphragm amplitude correlated positively with ex vivo force values and negatively with diaphragm fibrosis, a major cause of DMD muscle weakness. These studies validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in mdx diaphragm function in vivo. This technique will be valuable for testing potential therapies for DMD. ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease caused by dystrophin mutations. The mdx mouse is a widely used animal model of DMD. The mdx diaphragm muscle most closely recapitulates key features of DMD muscles, including progressive fibrosis and considerable force loss. Diaphragm function in mdx mice is commonly evaluated by specific force measurements ex vivo. While useful, this method only measures force from a small muscle sample at one time point. Therefore, accurate assessment of diaphragm function in vivo would provide an important advance to study the time course of functional decline and treatment benefits. Here, we evaluated an ultrasonography technique for measuring time-dependent changes of diaphragm function in mdx mice. Diaphragm movement amplitude values for mdx mice were considerably lower than those for wild-type, decreased from 8 to 18 months of age, and correlated strongly with ex vivo specific force. We then investigated the time course of diaphragm amplitude changes following administration of an adeno-associated viral vector expressing Flag-micro-dystrophin (AAV-µDys) to young adult mdx mice. Diaphragm amplitude peaked 4 weeks after AAV-µDys administration, and was 26% greater than control mdx mice at this time. This value decreased slightly to 21% above mdx controls after 12 weeks of treatment. Importantly, diaphragm amplitude again correlated strongly with ex vivo specific force. Also, diaphragm amplitude and specific force negatively correlated with fibrosis levels in the muscle. Together, our results validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in dystrophic diaphragm function in vivo, and for evaluating potential therapies for DMD.
Assuntos
Diafragma/diagnóstico por imagem , Diafragma/fisiopatologia , Distrofia Muscular Animal/diagnóstico por imagem , Distrofia Muscular Animal/fisiopatologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/diagnóstico por imagem , Distrofia Muscular de Duchenne/fisiopatologia , Reprodutibilidade dos Testes , UltrassonografiaRESUMO
The syntrophins alpha (SNTA) and beta 2 (SNTB2) are molecular adaptor proteins shown to stabilize ABCA1, an essential regulator of HDL cholesterol. Furthermore, SNTB2 is involved in glucose stimulated insulin release. Hyperglycemia and dyslipidemia are characteristic features of the metabolic syndrome, a serious public health problem with rising prevalence. Therefore, it is important to understand the role of the syntrophins herein. Mice deficient for both syntrophins (SNTA/B2-/-) have normal insulin and glucose tolerance, hepatic ABCA1 protein and cholesterol. When challenged with a HFD, wild type and SNTA/B2-/- mice have similar weight gain, adiposity, serum and liver triglycerides. Hepatic ABCA1, serum insulin and insulin sensitivity are normal while glucose tolerance is impaired. Liver cholesterol is reduced, and expression of SREBP2 and HMG-CoA-R is increased in the knockout mice. Scavenger receptor-BI (SR-BI) protein is strongly diminished in the liver of SNTA/B2-/- mice while SR-BI binding protein NHERF1 is not changed and PDZK1 is even induced. Knock-down of SNTA, SNTB2 or both has no effect on hepatocyte SR-BI and PDZK1 proteins. Further, SR-BI levels are not reduced in brown adipose tissue of SNTA/B2-/- mice excluding that syntrophins directly stabilize SR-BI. SR-BI stability is regulated by MAPK and phosphorylated ERK2 is induced in the liver of the knock-out mice. Blockage of ERK activity upregulates hepatocyte SR-BI showing that increased MAPK activity contributes to low SR-BI. Sphingomyelin which is well described to regulate cholesterol metabolism is reduced in the liver and serum of the knock-out mice while the size of serum lipoproteins is not affected. Current data exclude a major function of these syntrophins in ABCA1 activity and insulin release but suggest a role in regulating glucose uptake, ERK and SR-BI levels, and sphingomyelin metabolism in obesity.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Dieta Hiperlipídica , Proteínas Associadas à Distrofina/deficiência , Lipídeos/sangue , Fígado/metabolismo , Obesidade/metabolismo , Tecido Adiposo Marrom/metabolismo , Adiposidade , Animais , Glicemia/metabolismo , Linhagem Celular Tumoral , Colesterol/sangue , Modelos Animais de Doenças , Proteínas Associadas à Distrofina/genética , Ativação Enzimática , Genótipo , Intolerância à Glucose/sangue , Intolerância à Glucose/genética , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Obesidade/sangue , Obesidade/genética , Obesidade/fisiopatologia , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Receptores Depuradores Classe B/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Esfingomielinas/sangue , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Triglicerídeos/sangue , Aumento de PesoRESUMO
Alpha-syntrophin (SNTA) is an adaptor protein that regulates several signaling pathways. To analyze expression of SNTA immunoblot assays must be performed. Here, the specificity of four commercially available SNTA antibodies has been evaluated in immunoblot experiments using liver tissues of wild-type and SNTA-deficient mice. While one of the antibodies reacts with SNTA, two antibodies specifically recognize beta 2 syntrophin (SNTB2). The antigen detected by the fourth antibody has not been identified but is different from SNTA and SNTB2. Therefore, only one of the four tested antibodies is appropriate to analyze SNTA protein levels by immunoblot.
Assuntos
Especificidade de Anticorpos , Proteínas de Ligação ao Cálcio/imunologia , Immunoblotting , Proteínas de Membrana/imunologia , Proteínas Musculares/imunologia , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/metabolismo , Fígado/citologia , Fígado/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Musculares/deficiência , Proteínas Musculares/metabolismoRESUMO
Syntrophins are a family of cytoplasmic membrane-associated adaptor proteins, characterized by the presence of a unique domain organization comprised of a C-terminal syntrophin unique (SU) domain and an N-terminal pleckstrin homology (PH) domain that is split by insertion of a PDZ domain. Syntrophins have been recognized as an important component of many signaling events, and they seem to function more like the cell's own personal 'Santa Claus' that serves to 'gift' various signaling complexes with precise proteins that they 'wish for', and at the same time care enough for the spatial, temporal control of these signaling events, maintaining overall smooth functioning and general happiness of the cell. Syntrophins not only associate various ion channels and signaling proteins to the dystrophin-associated protein complex (DAPC), via a direct interaction with dystrophin protein but also serve as a link between the extracellular matrix and the intracellular downstream targets and cell cytoskeleton by interacting with F-actin. They play an important role in regulating the postsynaptic signal transduction, sarcolemmal localization of nNOS, EphA4 signaling at the neuromuscular junction, and G-protein mediated signaling. In our previous work, we reported a differential expression pattern of alpha-1-syntrophin (SNTA1) protein in esophageal and breast carcinomas. Implicated in several other pathologies, like cardiac dys-functioning, muscular dystrophies, diabetes, etc., these proteins provide a lot of scope for further studies. The present review focuses on the role of syntrophins in membrane targeting and regulation of cellular proteins, while highlighting their relevance in possible development and/or progression of pathologies including cancer which we have recently demonstrated.
Assuntos
Proteínas Associadas à Distrofina/metabolismo , Cromossomos/metabolismo , Proteínas Associadas à Distrofina/química , Humanos , Canais Iônicos/química , Canais Iônicos/metabolismo , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
α-Syntrophin is a component of the dystrophin scaffold-protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α-Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K(+) homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4-mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [(14)C]metabolites during sensory stimulation. Conscious KO and wild-type mice were pulse-labeled with [6-(14)C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer-assisted brain imaging or analysis of [(14)C]metabolites in extracts, respectively. High-resolution autoradiographic assays detected a 17% side-to-side difference (p < 0.05) in inferior colliculus of KO mice, not wild-type mice. However, there were no labeling differences between KO and wild-type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4-mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.
Assuntos
Aquaporina 4/metabolismo , Encéfalo/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Glucose/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Estimulação Acústica , Animais , Autorradiografia , Proteínas de Ligação ao Cálcio/deficiência , Radioisótopos de Carbono , Cromatografia Líquida de Alta Pressão , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/deficiência , Estimulação FísicaRESUMO
Adiponectin receptor 1 (AdipoR1) is one of the two signaling receptors of adiponectin with multiple beneficial effects in metabolic diseases. AdipoR1 C-terminal peptide is concordant with the consensus sequence of class I PSD-95, disc large, ZO-1 (PDZ) proteins, and screening of a liver yeast two hybrid library identified binding to ß2-syntrophin (SNTB2). Hybridization of a PDZ-domain array with AdipoR1 C-terminal peptide shows association with PDZ-domains of further proteins including ß1- and α-syntrophin (SNTA). Interaction of PDZ proteins and C-terminal peptides requires a free carboxy terminus next to the PDZ-binding region and is blocked by carboxy terminal added tags. N-terminal tagged AdipoR1 is more highly expressed than C-terminal tagged receptor suggesting that the free carboxy terminus may form a complex with PDZ proteins to regulate cellular AdipoR1 levels. The C- and N-terminal tagged AdipoR1 proteins are mainly localized in the cytoplasma. N-terminal but not C-terminal tagged AdipoR1 colocalizes with syntrophins in adiponectin incubated Huh7 cells. Adiponectin induced hepatic phosphorylation of AMPK and p38 MAPK which are targets of AdipoR1 is, however, not blocked in SNTA and SNTB2 deficient mice. Further, AdipoR1 protein is similarly abundant in the liver of knock-out and wild type mice when kept on a standard chow or a high fat diet. In summary these data suggest that AdipoR1 protein levels are regulated by so far uncharacterized class I PDZ proteins which are distinct from SNTA and SNTB2.
Assuntos
Proteínas Associadas à Distrofina/metabolismo , Hepatócitos/metabolismo , Domínios PDZ , Receptores de Adiponectina/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Linhagem Celular , Proteínas Associadas à Distrofina/química , Ativação Enzimática/fisiologia , Imunofluorescência , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases/metabolismo , Receptores de Adiponectina/química , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO-cGMP signalling with PDE5 inhibitors in dystrophin-deficient muscle.
Assuntos
Diafragma/efeitos dos fármacos , Fibrose/tratamento farmacológico , Debilidade Muscular/tratamento farmacológico , Distrofia Muscular de Duchenne/tratamento farmacológico , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Sulfonas/farmacologia , Animais , Creatina Quinase/sangue , GMP Cíclico/metabolismo , Diafragma/metabolismo , Diafragma/patologia , Modelos Animais de Doenças , Azul Evans/metabolismo , Fibrose/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular/efeitos dos fármacos , Fadiga Muscular/efeitos dos fármacos , Fadiga Muscular/fisiologia , Debilidade Muscular/etiologia , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/patologia , Óxido Nítrico/metabolismo , Purinas/farmacologia , Citrato de SildenafilaRESUMO
Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.
Assuntos
Dependovirus/genética , Distrofina/genética , Técnicas de Transferência de Genes , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/terapia , Músculos Respiratórios/fisiopatologia , Animais , Distrofina/uso terapêutico , Vetores Genéticos , Longevidade , Camundongos , Fibras Musculares Esqueléticas/fisiologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologiaRESUMO
α(1D)-Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α(1D)-ARs form a "signalosome" with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α(1D)-AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α(1D)-AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α(1D)-AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α(1D)-AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.
Assuntos
Complexo de Proteínas Associadas Distrofina/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais/fisiologia , alfa Catenina/metabolismo , Proteínas Associadas à Distrofina/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , RNA Interferente Pequeno/metabolismo , Receptores Adrenérgicos alfa 1/genética , alfa Catenina/genéticaRESUMO
α-Syntrophin (α-syn), a scaffold protein, links signaling molecules to the dystrophin-glycoprotein complex. Absence of α-syn from the DGC is known to lead to structurally aberrant neuromuscular junctions (NMJs) with few acetylcholine receptors (AChRs) clustered at synaptic sites. Using α-syn knock-out mice, we show that during the first postnatal week, α-syn is not required for synapse formation. However, at postnatal day 6 (P6)-P7, the structural integrity of the postsynaptic apparatus is altered, the turnover rate of AChRs increases significantly, and the number/density of AChRs is impaired. At the adult α-syn(-/-) NMJ, the turnover rate of AChRs is â¼ 4 times faster than wild-type synapses, and most removed receptors are targeted to degradation as few AChRs recycled to synaptic sites. Biochemical analyses show that in muscle cells of adult knock-out α-syn mice, total AChRs and scaffold protein rapsyn are significantly reduced, the 89 kDa and 75 kDa isoforms of tyrosine phosphorylated α-dystrobrevin (α-dbn) 1 (which are required for the maintenance and stability of AChR in α-dbn(-/-) synapses) are barely detectable. Electroporation of GFP-α-dbn1 in α-syn(-/-) muscle cells partially restored receptor density, turnover rate, and the structural integrity of the postsynaptic apparatus, whereas expression of rapsyn-GFP failed to rescue the α-syn(-/-) synaptic phenotype. These results demonstrate that α-syn is required for the maturation and stability of the postsynaptic apparatus and suggest that α-syn may act via α-dbn1.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/deficiência , Proteínas Musculares/deficiência , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/metabolismo , Receptores Nicotínicos/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Bungarotoxinas/farmacocinética , Eletroporação/métodos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrazinas/farmacocinética , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Transporte Proteico/genética , RNA Mensageiro/metabolismo , Receptores Nicotínicos/genéticaRESUMO
Expression of the water channel aquaporin-4 (AQP4) at the blood-brain interface is dependent upon the dystrophin associated protein complex. Here we investigated whether deletion of the Aqp4 gene affects the molecular composition of this protein scaffold and the integrity of the blood-brain barrier. High-resolution immunogold cytochemistry revealed that perivascular expression of α-syntrophin was reduced by 60% in Aqp4(-/-) mice. Additionally, perivascular AQP4 expression was reduced by 88% in α-syn(-/-) mice, in accordance with earlier reports. Immunofluorescence showed that Aqp4 deletion also caused a modest reduction in perivascular dystrophin, whereas ß-dystroglycan labeling was unaltered. Perivascular microglia were devoid of AQP4 immunoreactivity. Deletion of Aqp4 did not alter the ultrastructure of capillary endothelial cells, the expression of tight junction proteins (claudin-5, occludin, and zonula occludens 1), or the vascular permeability to horseradish peroxidase and Evans blue albumin dye. We conclude that Aqp4 deletion reduces the expression of perivascular glial scaffolding proteins without affecting the endothelial barrier. Our data also indicate that AQP4 and α-syntrophin are mutually dependent upon each other for proper perivascular expression.
Assuntos
Aquaporina 4/deficiência , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Endotélio/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Neuroglia/metabolismo , Animais , Aquaporina 4/genética , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/ultraestrutura , Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade Capilar/genética , Córtex Cerebral/citologia , Endotélio/ultraestrutura , Azul Evans , Regulação da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microscopia Imunoeletrônica , Proteínas Musculares/metabolismo , Neuroglia/ultraestrutura , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
Syntrophins are adaptor proteins that link intracellular signaling molecules to the dystrophin based scaffold. In this study, we investigated the function of syntrophins in cell migration, one of the early steps in myogenic differentiation and in regeneration of adult muscle. Hepatocyte growth factor (HGF) stimulates migration and lamellipodia formation in cultured C2 myoblasts. In the migrating cells, syntrophin concentrated in the rear-lateral region of the cell, opposite of the lamellipodia, instead of being diffusely present throughout the cytoplasm of non-migrating cells. When the expression of α-syntrophin, the major syntrophin isoform of skeletal muscle, was reduced by transfection with the α-syntrophin-specific siRNA, HGF stimulation of lamellipodia formation was prevented. Likewise, migration of myoblasts from α-syntrophin knockout mice could not be stimulated by HGF. However, HGF-induced migration was restored in myoblasts isolated from a transgenic mouse expressing α-syntrophin only in muscle cells. Treatment of C2 myoblasts with inhibitors of PI3-kinase not only reduced the rate of cell migration, but also impaired the accumulation of syntrophins in the rear-lateral region of the migrating cells. Phosphorylation of Akt was reduced in the α-syntrophin siRNA-treated C2 cells. These results suggest that α-syntrophin is required for HGF-induced migration of myoblasts and for proper PI3-kinase/Akt signaling.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/fisiologia , Fator de Crescimento de Hepatócito/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Mioblastos/citologia , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Musculares/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/genética , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/metabolismo , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
At the neuromuscular junction (NMJ), the dystrophin protein complex provides a scaffold that functions to stabilize acetylcholine receptor (AChR) clusters. Syntrophin, a key component of that scaffold, is a multidomain adapter protein that links a variety of signaling proteins and ion channels to the dystrophin protein complex. Without syntrophin, utrophin and neuronal nitric oxide synthase mu (nNOSmu) fail to localize to the NMJ and the AChRs are distributed abnormally. Here we investigate the contribution of syntrophin domains to AChR distribution and to localization of utrophin and nNOSmu at the NMJ. Transgenic mice expressing alpha-syntrophin lacking portions of the first pleckstrin homology (PH) domain (DeltaPH1a or DeltaPH1b) or the entire PDZ domain (DeltaPDZ) were bred onto the alpha-syntrophin null background. As expected the DeltaPDZ transgene did not restore the NMJ localization of nNOS. The DeltaPH1a transgene did restore postsynaptic nNOS but surprisingly did not restore sarcolemmal nNOS (although sarcolemmal aquaporin-4 was restored). Mice lacking the alpha-syntrophin PDZ domain or either half of the PH1 domain were able to restore utrophin to the NMJ but did not correct the aberrant AChR distribution of the alpha-syntrophin knock-out mice. However, mice expressing both the transgenic DeltaPDZ and the transgenic DeltaPH1a constructs did restore normal AChR distribution, demonstrating that both domains are required but need not be confined within the same protein to function. We conclude that the PH1 and PDZ domains of alpha-syntrophin work in concert to facilitate the localization of AChRs and nNOS at the NMJ.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Junção Neuromuscular/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Receptores Colinérgicos/metabolismo , Utrofina/metabolismo , Animais , Aquaporina 4/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Domínios PDZ , RNA Mensageiro/metabolismo , Sarcolema/metabolismoRESUMO
α(1D)-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α(1D)-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, ß(1) and ß(2)) interact with α(1D)-ARs and our previous studies suggest multiple isoforms are required for proper α(1D)-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α(1D)-AR function. Radioligand binding experiments reveal α-syntrophin enhances α(1D)-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate ß(2)-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α(1D)-AR/DAPC signalosome significantly alter the pharmacological properties of α(1)-AR ligands in vitro.