RESUMO
Muscular dystrophy is a group of genetic disorders that lead to muscle wasting and loss of muscle function. Identifying genetic modifiers that alleviate symptoms or enhance the severity of a primary disease helps to understand mechanisms behind disease pathology and facilitates discovery of molecular targets for therapy. Several muscular dystrophies are caused by genetic defects in the components of the dystrophin-glycoprotein adhesion complex (DGC). Thrombospondin-4 overexpression has been shown to mitigate dystrophic disease in mouse models for Duchenne muscular dystrophy (dystrophin deficiency) and limb-girdle muscular dystrophy type 2F (LGMD2F, δ-sarcoglycan deficiency), while deletion of the thrombospondin-4 gene exacerbated the diseases. Hence, thrombospondin-4 has been considered a candidate molecule for therapy of muscular dystrophies involving the DGC. We have investigated whether thrombospondin-4 could act as a genetic modifier for other DGC-associated diseases: limb-girdle muscular dystrophy type 2E (LGMD2E, ß-sarcoglycan deficiency) and laminin α2 chain-deficient muscular dystrophy (LAMA2-RD). Deletion of the thrombospondin-4 gene in mouse models for LGMD2E and LAMA2-RD, respectively, did not result in worsening of the dystrophic phenotype. Loss of thrombospondin-4 did not enhance sarcolemma damage and did not impair trafficking of transmembrane receptors integrin α7ß1 and dystroglycan in double knockout muscles. Our results suggest that thrombospondin-4 might not be a relevant therapeutic target for all muscular dystrophies involving the DGC. This data also demonstrates that molecular pathology between very similar diseases like LGMD2E and 2F can differ significantly.
Assuntos
Laminina , Camundongos Knockout , Sarcoglicanas , Trombospondinas , Animais , Laminina/metabolismo , Laminina/genética , Laminina/deficiência , Sarcoglicanas/genética , Sarcoglicanas/deficiência , Sarcoglicanas/metabolismo , Camundongos , Trombospondinas/genética , Trombospondinas/metabolismo , Trombospondinas/deficiência , Modelos Animais de Doenças , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Deleção de Genes , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologiaRESUMO
Muscular dystrophies in dogs and cats represent a heterogeneous group of inherited, sometimes congenital, but infrequently diagnosed, progressive neuromuscular disorders. A correct identification and characterization of canine and feline muscular dystrophies could increase diagnostic and treatment strategies for veterinary neurologists and could identify useful animal models for the study of human dystrophies. However, in dogs and cats, diagnosis of muscular dystrophies is challenging due to a nonspecific clinical phenotype and pathological lesions, thus is most likely underestimated. We performed immunofluorescence and Western blot techniques using a wide panel of antibodies against proteins involved in human dystrophies (dystrophin mid-rod and carboxyterminal domain, α, ß, γ, and δ-sarcoglycan, α-dystroglycan, caveolin-3, emerin, merosin, dysferlin, calpain-3, spectrin epitopes), on 9 canine and 3 feline muscle biopsies characterized by myopathic changes. Dystrophin deficiency was detected in 3 dogs and 2 novel canine muscular dystrophies have been identified, characterized by deficiency of caveolin-3 and calpain-3, respectively. In 2 cats, deficiency of ß-SG and carboxyterminal domain of dystrophin in all muscle fibers has been detected. Performing immunofluorescence and Western blot analyses with a wider panel of antibodies allowed a correct identification of muscular dystrophies in dogs and cats and provides a direction for subsequent targeted genetic testing.
Assuntos
Doenças do Gato , Doenças do Cão , Distrofina/metabolismo , Distrofias Musculares/metabolismo , Sarcoglicanas/genética , Animais , Gatos , Cães , Imuno-Histoquímica/veterinária , Músculo Esquelético , Distrofias Musculares/genética , Distrofias Musculares/patologia , Sarcoglicanas/análise , Sarcoglicanas/deficiênciaRESUMO
Genetic cardiomyopathy is a group of intractable cardiovascular disorders involving heterogeneous genetic contribution. This heterogeneity has hindered the development of life-saving therapies for this serious disease. Genetic mutations in dystrophin and its associated glycoproteins cause cardiomuscular dysfunction. Large animal models incorporating these genetic defects are crucial for developing effective medical treatments, such as tissue regeneration and gene therapy. In the present study, we knocked out the δ-sarcoglycan (δ-SG) gene (SGCD) in domestic pig by using a combination of efficient de novo gene editing and somatic cell nuclear transfer. Loss of δ-SG expression in the SGCD knockout pigs caused a concomitant reduction in the levels of α-, ß-, and γ-SG in the cardiac and skeletal sarcolemma, resulting in systolic dysfunction, myocardial tissue degeneration, and sudden death. These animals exhibited symptoms resembling human genetic cardiomyopathy and are thus promising for use in preclinical studies of next-generation therapies.
Assuntos
Cardiomiopatias , Sarcoglicanas , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Feminino , Mutação da Fase de Leitura/genética , Técnicas de Inativação de Genes , Masculino , Miocárdio/química , Miocárdio/metabolismo , Miocárdio/patologia , Sarcoglicanas/deficiência , Sarcoglicanas/genética , SuínosRESUMO
Muscular Dystrophies are severe genetic diseases due to mutations in structural genes, characterized by progressive muscle wasting that compromises patients' mobility and respiratory functions. Literature underlined oxidative stress and inflammation as key drivers of these pathologies. Interestingly among different myofiber classes, type I fibers display a milder dystrophic phenotype showing increased oxidative metabolism. This work shows the benefits of a cyanidin-enriched diet, that promotes muscle fiber-type switch and reduced inflammation in dystrophic alpha-sarcoglyan (Sgca) null mice having, as a net outcome, morphological and functional rescue. Notably, this benefit is achieved also when the diet is administered in dystrophic animals when the signs of the disease are seriously evident. Our work provides compelling evidence that a cyanidin-rich diet strongly delays the progression of muscular dystrophies, paving the way for a combinatorial approach where nutritional-based reduction of muscle inflammation and oxidative stress facilitate the successful perspectives of definitive treatments.
Assuntos
Antocianinas/administração & dosagem , Suplementos Nutricionais , Mediadores da Inflamação/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Sarcoglicanopatias/dietoterapia , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Masculino , Camundongos Knockout , Mitocôndrias Musculares/patologia , Músculo Esquelético/patologia , Biogênese de Organelas , Fenótipo , Carbonilação Proteica , Sarcoglicanopatias/genética , Sarcoglicanopatias/metabolismo , Sarcoglicanopatias/patologia , Sarcoglicanas/deficiência , Sarcoglicanas/genéticaRESUMO
BACKGROUND: Metformin is a popular antidiabetic agent that is also used to treat heart failure patients with type 2 diabetes mellitus. Several reports suggest that metformin may also have cardioprotective effects in patients without diabetes mellitus. In the present study, we investigated the possible therapeutic effect of metformin in heart failure and its underlying molecular mechanisms using a δ-sarcoglycan-deficient mouse model of dilated cardiomyopathy. METHODS AND RESULTS: Thirty-two-week-old δ-sarcoglycan-deficient mice exhibiting established cardiomyopathy with extensive left ventricular dilatation and dysfunction were administered saline or metformin (200 mg/kg per day) for 4 weeks using osmotic mini-pumps. Metformin partially reversed the left ventricular dilatation (reverse remodeling) and significantly improved cardiac function. The hearts of metformin-treated mice showed less fibrosis, less cardiomyocyte hypertrophy, and fewer degenerative subcellular changes than saline-treated mice. These effects were accompanied by restored expression of the sarcomeric proteins myosin heavy chain and troponin I, and their transcription factor, GATA-4. Autophagy was enhanced in the hearts from metformin-treated mice, as indicated by increase of myocardial microtubule-associated protein-1 LC-3 (light chain 3)-II levels and LC3-II/-I ratios as well as levels of cathepsin D and ATP. In addition, increased numbers of autophagic vacuoles and lysosomes were accompanied increased AMP-activated protein kinase activity and suppression of mammalian target of rapamycin phosphorylation. Finally, autophagic flux assays using short-term chloroquine treatment revealed that autophagy was activated in δ-sarcoglycan-deficient hearts and was further augmented by metformin treatment. CONCLUSIONS: Metformin is a beneficial pharmacological tool that mitigates heart failure caused by δ-sarcoglycan deficiency in association with enhanced autophagy.
Assuntos
Autofagia/fisiologia , Cardiomiopatias/genética , Sarcoglicanas/deficiência , Remodelação Ventricular/genética , Animais , Autofagia/genética , Cardiomegalia/metabolismo , Cardiomiopatias/metabolismo , Cardiomiopatia Dilatada/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insuficiência Cardíaca/genética , Hipoglicemiantes/uso terapêutico , Metformina/farmacologia , Camundongos Transgênicos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/efeitos dos fármacosRESUMO
Galectin-3 is a ß-galactoside-binding lectin which is important in cell proliferation and apoptotic regulation. Recently, serum galectin-3 has been shown to have prognostic value as a biomarker in heart failure. Encephalomyocarditis virus (EMCV) can cause severe myocarditis, congestive heart failure and dilated cardiomyopathy as well as encephalitis in various animals including mice. The pathophysiological role of galectin-3 in acute myocarditis following viral infection is not fully understood. The goal of this study is to determine the cardiac localization and the time-course of galectin-3 expression in heart failure after viral inoculation with EMCV. At 12, 24, 48, 96 hours, 7 and 10 days after intraperitoneal EMCV inoculation, animals were examined histologically and analyzed for the expression of galectin-3 and Iba1. Galectin-3 was up-regulated in degenerated fibrotic lesions of cardiac tissues 96 hours after viral inoculation and were followed by myocardial fibrosis. At the same time, Iba1 positive macrophages were observed within the inflammatory sites. A time-course correlation between the number of galectin-3 positive cells and the cardiac area of degenerated fibrotic lesions was detected-serum galectin-3 increased at 96 hours and correlated well with the number of cardiac galectin-3 positive cells. Our results indicate that galectin-3 expression may be a useful biomarker of cardiac fibrotic degeneration in acute myocarditis following viral infection. In addition, measuring serum galectin-3 levels might be an early diagnostic method for detecting cardiac degeneration in acute myocarditis.
Assuntos
Infecções por Cardiovirus/sangue , Infecções por Cardiovirus/metabolismo , Vírus da Encefalomiocardite , Galectina 3/sangue , Galectina 3/metabolismo , Miocardite/sangue , Miocardite/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatia Dilatada/sangue , Cardiomiopatia Dilatada/metabolismo , Infecções por Cardiovirus/patologia , Modelos Animais de Doenças , Vírus da Encefalomiocardite/patogenicidade , Fibrose , Imuno-Histoquímica , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Miocardite/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Prognóstico , Sarcoglicanas/deficiência , Sarcoglicanas/genéticaRESUMO
In skeletal muscle, extracellular matrix (ECM) remodeling can either support the complete regeneration of injured muscle or facilitate pathologic fibrosis and muscle degeneration. Muscular dystrophy (MD) is a group of genetic disorders that results in a progressive decline in muscle function and is characterized by the abundant deposition of fibrotic tissue. Unlike acute injury, where ECM remodeling is acute and transient, in MD, remodeling persists until fibrosis obstructs the regenerative efforts of diseased muscles. Thus, understanding how ECM is deposited and organized is critical in the context of muscle repair. Connective tissue growth factor (CTGF or CCN2) is a matricellular protein expressed by multiple cell types in response to tissue injury. Although used as a general marker of fibrosis, the cell type-dependent role of CTGF in dystrophic muscle has not been elucidated. To address this question, a conditional Ctgf myofiber and fibroblast-knockout mouse lines were generated and crossed to a dystrophic background. Only myofiber-selective inhibition of CTGF protected δ-sarcoglycan-null ( Sgcd-/-) mice from the dystrophic phenotype, and it did so by affecting collagen organization in a way that allowed for improvements in dystrophic muscle regeneration and function. To confirm that muscle-specific CTGF functions to mediate collagen organization, we generated mice with transgenic muscle-specific overexpression of CTGF. Again, genetic modulation of CTGF in muscle was not sufficient to drive fibrosis, but altered collagen content and organization after injury. Our results show that the myofibers are critical mediators of the deleterious effects associated with CTGF in MD and acutely injured skeletal muscle.-Petrosino, J. M., Leask, A., Accornero, F. Genetic manipulation of CCN2/CTGF unveils cell-specific ECM-remodeling effects in injured skeletal muscle.
Assuntos
Fator de Crescimento do Tecido Conjuntivo , Matriz Extracelular , Regulação da Expressão Gênica , Fibras Musculares Esqueléticas , Distrofia Muscular Animal , Animais , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fator de Crescimento do Tecido Conjuntivo/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrose , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Sarcoglicanas/deficiênciaRESUMO
In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.
Assuntos
Trifosfato de Adenosina/imunologia , Distrofia Muscular Animal , Miofibrilas , Sarcoglicanas/deficiência , Linfócitos T Reguladores , Trifosfato de Adenosina/genética , Animais , Cálcio/imunologia , Doença Crônica , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/imunologia , Distrofia Muscular Animal/patologia , Miofibrilas/imunologia , Miofibrilas/patologia , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/imunologia , Sarcoglicanas/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
OBJECTIVES: We hypothesized that systemic administration of high-mobility group box 1 fragment attenuates the progression of myocardial fibrosis and cardiac dysfunction in a hamster model of dilated cardiomyopathy by recruiting bone marrow mesenchymal stem cells thus causing enhancement of a self-regeneration system. METHODS: Twenty-week-old J2N-k hamsters, which are δ-sarcoglycan-deficient, were treated with systemic injection of high-mobility group box 1 fragment (HMGB1, n = 15) or phosphate buffered saline (control, n = 11). Echocardiography for left ventricular function, cardiac histology, and molecular biology were analyzed. The life-prolonging effect was assessed separately using the HMGB1 and control groups, in addition to a monthly HMGB1 group which received monthly systemic injections of high-mobility group box 1 fragment, 3 times (HMGB1, n = 11, control, n = 9, monthly HMGB1, n = 9). RESULTS: The HMGB1 group showed improved left ventricular ejection fraction, reduced myocardial fibrosis, and increased capillary density. The number of platelet-derived growth factor receptor-alpha and CD106 positive mesenchymal stem cells detected in the myocardium was significantly increased, and intra-myocardial expression of tumor necrosis factor α stimulating gene 6, hepatic growth factor, and vascular endothelial growth factor were significantly upregulated after high-mobility group box 1 fragment administration. Improved survival was observed in the monthly HMGB1 group compared with the control group. CONCLUSIONS: Systemic high-mobility group box 1 fragment administration attenuates the progression of left ventricular remodeling in a hamster model of dilated cardiomyopathy by enhanced homing of bone marrow mesenchymal stem cells into damaged myocardium, suggesting that high-mobility group box 1 fragment could be a new treatment for dilated cardiomyopathy.
Assuntos
Células da Medula Óssea/metabolismo , Cardiomiopatia Dilatada , Autorrenovação Celular , Proteína HMGB1/farmacologia , Células-Tronco Mesenquimais/metabolismo , Sarcoglicanas/deficiência , Função Ventricular Esquerda , Animais , Animais Geneticamente Modificados , Células da Medula Óssea/patologia , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Cricetinae , Modelos Animais de Doenças , Ecocardiografia , Fibrose , Proteína HMGB1/genética , Células-Tronco Mesenquimais/patologia , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/genéticaRESUMO
Generation of superoxide by xanthine oxidase can be stimulated under ischemic and aberrant calcium homeostasis. Because patients and mice with Duchenne muscular dystrophy (DMD) suffer from ischemia and excessive calcium influx, we tested the hypothesis that xanthine oxidase activity is elevated and contributes to disease pathology. Xanthine oxidase activity was measured by urinary isoxanthopterin in DMD patients at rest and in response to exercise. Urinary isoxanthopterin/creatinine was elevated compared to age-matched controls and Becker muscular dystrophy (BMD) patients. Concentrations were also increased after a six minute walk test in ambulatory patients. We also measured urinary isoxanthopterin in wildtype mice and a number of dystrophic mouse models; the DMD mouse model (mdx), mdx mice overexpressing a variety of transgenic miniaturized and chimeric skeletal muscle-specific dystrophins and utrophin and the ß-sarcoglycan deficient (Scgb-/-) mouse which represents type 2E human limb-girdle muscular dystrophy. Mdx and Scgb-/-mice had greater urinary isoxanthopterin/creatinine than wildtype mice while mdx mice expressing dystrophin or utrophin linking the extracellular matrix to the actin cytoskeleton were not different than wildtype. We also measured higher levels of urinary ortho-tyrosine in humans and mice deficient for dystrophin to confirm elevated oxidative stress. Surprisingly, mdx had lower xanthine oxidase protein levels and higher mRNA in gastrocnemius muscle compared to wildtype mice, however, the enzymatic activity of skeletal muscle xanthine oxidase was elevated above wildtype and a transgenic rescued mdx mouse (DysΔMTB-mdx). Downhill treadmill running also caused significant increases in mdx urinary isoxanthopterin that was prevented with the xanthine oxidase inhibitor allopurinol. Similarly, in vitro eccentric contraction-induced force drop of mdx muscle was attenuated by the allopurinol metabolite, oxypurinol. Together, our data suggests hyper-activity of xanthine oxidase in DMD, identifies xanthine oxidase activity as a contributing factor in eccentric contraction-induced force drop of dystrophin-deficient skeletal muscle and highlights the potential of isoxanthopterin as a noninvasive biomarker in DMD.
Assuntos
Distrofina/deficiência , Distrofia Muscular Animal/enzimologia , Distrofia Muscular de Duchenne/enzimologia , Xantina Oxidase/urina , Xantopterina/urina , Adolescente , Alopurinol/farmacologia , Animais , Biomarcadores/urina , Estudos de Casos e Controles , Creatinina/urina , Distrofina/genética , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Oxipurinol/farmacologia , Sarcoglicanas/deficiência , Sarcoglicanas/genética , Tirosina/urina , Utrofina/deficiência , Utrofina/genética , Xantina Oxidase/genética , Adulto JovemRESUMO
BACKGROUND AND AIMS: Several studies propose that (-)-epicatechin, a flavonol present in high concentration in the cocoa, has cardioprotective effects. This study aimed to evaluate the impact of (-)-epicatechin on the development of dilated cardiomyopathy in a δ sarcoglycan null mouse model. METHODS AND RESULTS: δ Sarcoglycan null mice were treated for 15 days with (-)-epicatechin. Histological and morphometric analysis of the hearts treated mutant mice showed significant reduction of the vasoconstrictions in the coronary arteries as well as fewer areas with fibrosis and a reduction in the loss of the ventricular wall. On the contrary, it was observed a thickening of this region. By Western blot analysis, it was shown, and increment in the phosphorylation level of eNOS and PI3K/AKT/mTOR/p70S6K proteins in the heart of the (-)-epicatechin treated animals. On the other hand, we observed a significantly decreased level of the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) heart failure markers. CONCLUSION: All the results indicate that (-)-epicatechin has the potential to prevent the development of dilated cardiomyopathy of genetic origin and encourages the use of this flavonol as a pharmacological therapy for dilated cardiomyopathy and heart failure diseases.
Assuntos
Cardiomiopatia Dilatada/prevenção & controle , Catequina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Sarcoglicanas/deficiência , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Fator Natriurético Atrial/metabolismo , Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/enzimologia , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Fibrose , Masculino , Camundongos Knockout , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Peptídeo Natriurético Encefálico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sarcoglicanas/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
Cell engraftment, survival and integration during transplantation procedures represent the crux of cell-based therapies. Thus, there have been many studies focused on improving cell viability upon implantation. We used severe oxidative stress to select for a mouse mesoangioblast subpopulation in vitro and found that this subpopulation retained self-renewal and myogenic differentiation capacities while notably enhancing cell survival, proliferation and migration relative to unselected cells. Additionally, this subpopulation of cells presented different resistance and recovery properties upon oxidative stress treatment, demonstrating select advantages over parental mesoangioblasts in our experimental analysis. Specifically, the cells were resistant to oxidative environments, demonstrating survival, continuous self-renewal and improved migration capability. The primary outcome of the selected cells was determined in in vivo experiments in which immunocompromised dystrophic mice were injected intramuscularly in the tibialis anterior with selected or non-selected mesoangioblasts. Resistant mesoangioblasts exhibited markedly enhanced survival and integration into the host skeletal muscle, accounting for a more than 70% increase in engraftment compared with that of the unselected mesoangioblast cell population and leading to remarkable muscle recovery. Thus, the positive effects of sorting on mesoangioblast cell behaviour in vitro and in vivo suggest that a selection step involving oxidative stress preconditioning may provide a novel methodology to select for resistant cells for use in regenerative tissue applications to prevent high mortality rates upon transplantation.
Assuntos
Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos SCID , Músculo Esquelético/citologia , Distrofia Muscular Animal/terapia , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sarcoglicanas/deficiência , Sarcoglicanas/genética , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVES: To characterise the pattern and spectrum of involvement on muscle MRI in a large cohort of patients with sarcoglycanopathies, which are limb-girdle muscular dystrophies (LGMD2C-2F) caused by mutations in one of the four genes coding for muscle sarcoglycans. METHODS: Lower limb MRI scans of patients with LGMD2C-2F, ranging from severe childhood variants to milder adult-onset forms, were collected in 17 neuromuscular referral centres in Europe and USA. Muscle involvement was evaluated semiquantitatively on T1-weighted images according to a visual score, and the global pattern was assessed as well. RESULTS: Scans from 69 patients were examined (38 LGMD2D, 18 LGMD2C, 12 LGMD2E and 1 LGMD2F). A common pattern of involvement was found in all the analysed scans irrespective of the mutated gene. The most and earliest affected muscles were the thigh adductors, glutei and posterior thigh groups, while lower leg muscles were relatively spared even in advanced disease. A proximodistal gradient of involvement of vasti muscles was a consistent finding in these patients, including the most severe ones. CONCLUSIONS: Muscle involvement on MRI is consistent in patients with LGMD2C-F and can be helpful in distinguishing sarcoglycanopathies from other LGMDs or dystrophinopathies, which represent the most common differential diagnoses. Our data provide evidence about selective susceptibility or resistance to degeneration of specific muscles when one of the sarcoglycans is deficient, as well as preliminary information about progressive involvement of the different muscles over time.
Assuntos
Imageamento por Ressonância Magnética/métodos , Sarcoglicanopatias/genética , Sarcoglicanas/genética , Adolescente , Adulto , Criança , Pré-Escolar , Europa (Continente) , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Mutação , Fenótipo , Sarcoglicanas/deficiência , Estados UnidosRESUMO
BACKGROUND: Smooth muscle cells are important for atherosclerotic plaque stability. Their proper ability to communicate with the extracellular matrix is crucial for maintaining the correct tissue integrity. In this study, we have investigated the role of ß-sarcoglycan within the matrix-binding dystrophin-glycoprotein complex in the development of atherosclerosis. RESULTS: Atherosclerotic plaque development was significantly reduced in ApoE-deficient mice lacking ß-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking ß-sarcoglycan showed a reduction in ovarian adipose tissue and adipocyte size, while the total weight of the animals was not significantly different. Western blot analysis of adipose tissues showed a decreased activation of protein kinase B, while that of AMP-activated kinase was increased in mice lacking ß-sarcoglycan. Analysis of plasma in ß-sarcoglycan-deficient mice revealed reduced levels of leptin, adiponectin, insulin, cholesterol, and triglycerides but increased levels of IL-6, IL-17, and TNF-α. CONCLUSIONS: Our results indicate that the dystrophin-glycoprotein complex and ß-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of ß-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction.
Assuntos
Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica , Sarcoglicanas/deficiência , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Complexo de Proteínas Associadas Distrofina/metabolismo , Feminino , Predisposição Genética para Doença , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sarcoglicanas/genéticaRESUMO
Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.
Assuntos
Complexo de Proteínas Associadas Distrofina/química , Terapia Genética , Distrofia Muscular do Cíngulo dos Membros/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Engenharia de Proteínas , Sarcoglicanas/genética , Animais , Códon sem Sentido/genética , Diafragma/metabolismo , Diafragma/patologia , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Éxons , Fibrose , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/terapia , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Oligonucleotídeos Antissenso/farmacologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Sarcoglicanas/biossíntese , Sarcoglicanas/química , Sarcoglicanas/deficiência , Sarcolema/metabolismo , Deleção de SequênciaRESUMO
Muscular dystrophy (MD) is a disease characterized by skeletal muscle necrosis and the progressive accumulation of fibrotic tissue. While transforming growth factor (TGF)-ß has emerged as central effector of MD and fibrotic disease, the cell types in diseased muscle that underlie TGFß-dependent pathology have not been segregated. Here, we generated transgenic mice with myofiber-specific inhibition of TGFß signaling owing to expression of a TGFß type II receptor dominant-negative (dnTGFßRII) truncation mutant. Expression of dnTGFßRII in myofibers mitigated the dystrophic phenotype observed in δ-sarcoglycan-null (Sgcd(-/-)) mice through a mechanism involving reduced myofiber membrane fragility. The dnTGFßRII transgene also reduced muscle injury and improved muscle regeneration after cardiotoxin injury, as well as increased satellite cell numbers and activity. An unbiased global expression analysis revealed a number of potential mechanisms for dnTGFßRII-mediated protection, one of which was induction of the antioxidant protein metallothionein (Mt). Indeed, TGFß directly inhibited Mt gene expression in vitro, the dnTGFßRII transgene conferred protection against reactive oxygen species accumulation in dystrophic muscle and treatment with Mt mimetics protected skeletal muscle upon injury in vivo and improved the membrane stability of dystrophic myofibers. Hence, our results show that the myofibers are central mediators of the deleterious effects associated with TGFß signaling in MD.
Assuntos
Distrofias Musculares/genética , Miofibrilas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/patologia , Proteínas Cardiotóxicas de Elapídeos/farmacologia , Crotoxina/farmacologia , Modelos Animais de Doenças , Combinação de Medicamentos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Metalotioneína/genética , Metalotioneína/metabolismo , Camundongos , Camundongos Transgênicos , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Mutação , Miofibrilas/efeitos dos fármacos , Miofibrilas/patologia , Proteínas Serina-Treonina Quinases/deficiência , Espécies Reativas de Oxigênio/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Sarcoglicanas/deficiência , Sarcoglicanas/genética , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Fator de Crescimento Transformador beta/farmacologia , TransgenesRESUMO
Disruption of the dystrophin complex causes muscle injury, dysfunction, cell death and fibrosis. Excess transforming growth factor (TGF) ß signaling has been described in human muscular dystrophy and animal models, where it is thought to relate to the progressive fibrosis that characterizes dystrophic muscle. We now found that canonical TGFß signaling acutely increases when dystrophic muscle is stimulated to contract. Muscle lacking the dystrophin-associated protein γ-sarcoglycan (Sgcg null) was subjected to a lengthening protocol to produce maximal muscle injury, which produced rapid accumulation of nuclear phosphorylated SMAD2/3. To test whether reducing SMAD signaling improves muscular dystrophy in mice, we introduced a heterozygous mutation of SMAD4 (S4) into Sgcg mice to reduce but not ablate SMAD4. Sgcg/S4 mice had improved body mass compared with Sgcg mice, which normally show a wasting phenotype similar to human muscular dystrophy patients. Sgcg/S4 mice had improved cardiac function as well as improved twitch and tetanic force in skeletal muscle. Functional enhancement in Sgcg/S4 muscle occurred without a reduction in fibrosis, suggesting that intracellular SMAD4 targets may be important. An assessment of genes differentially expressed in Sgcg muscle focused on those encoding calcium-handling proteins and responsive to TGFß since this pathway is a target for mediating improvement in muscular dystrophy. These data demonstrate that excessive TGFß signaling alters cardiac and muscle performance through the intracellular SMAD pathway.
Assuntos
Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Miocárdio/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Peso Corporal , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Testes de Função Cardíaca , Humanos , Proteínas de Ligação a TGF-beta Latente/deficiência , Proteínas de Ligação a TGF-beta Latente/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Mutação , Miocárdio/patologia , Fosforilação , Sarcoglicanas/deficiência , Sarcoglicanas/genética , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: The sarcoglycan alpha gene, also known as the adhalin gene, is located on chromosome 17q21; mutations in this gene are associated with limb-girdle muscular dystrophy type 2D. We describe two Turkish siblings with findings consistent with limb-girdle muscular dystrophy type 2D. The evaluation excluded a dystrophinopathy, which is the most common form of muscular dystrophy. PATIENTS: Both siblings had very high levels of creatinine phosphokinase and negative molecular tests for deletions and duplications of the dystrophin gene. The older boy presented at 8 years of age with an inability to climb steps and an abnormal gait. His younger brother was 5 years old and had similar symptoms. The muscle biopsy evaluation was performed only in the older brother. RESULTS: The muscle biopsy showed dystrophic features as well as a deficiency in the expression of two different glycoproteins: the alpha sarcoglycan and the gamma sarcoglycan. Sarcolemmal expressions of dystrophin and other sarcoglycans (beta and delta) were diffusely present. DNA analysis demonstrated the presence of previously unknown homozygous mutations [c.226 C > T (p.L76 F)] in exon 3 in the sarcoglycan alpha genes of both siblings. Similar heterozygous point mutations at the same locus were found in both parents, but the genes of beta, delta, and gamma sarcoglycan were normal in the remaining family members. CONCLUSIONS: We describe two siblings with limb-girdle muscular dystrophy type 2D with a novel missense mutation. These patients illustrate that the differential diagnosis of muscular dystrophies is impossible with clinical findings alone. Therefore, a muscle biopsy and DNA analysis remain essential methods for diagnosis of muscle diseases.
Assuntos
Mutação de Sentido Incorreto , Sarcoglicanopatias/genética , Sarcoglicanopatias/fisiopatologia , Sarcoglicanas/deficiência , Sarcoglicanas/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Análise Mutacional de DNA , Diagnóstico Diferencial , Pai , Humanos , Masculino , Dados de Sequência Molecular , Mães , Músculo Esquelético/patologia , Sarcoglicanopatias/diagnóstico , Sarcoglicanopatias/patologia , Irmãos , TurquiaRESUMO
The loss of dystrophin or its associated proteins results in the development of muscle wasting frequently associated with cardiomyopathy. Contractile cardiac tissue is injured and replaced by fibrous tissue or fatty infiltrates, leading to a progressive decrease of the contractile force and finally to end-stage heart failure. At the time symptoms appear, restoration of a functional allele of the causative gene might not be sufficient to prevent disease progression. Alterations in Ca(2+) transport and intracellular calcium levels have been implicated in many types of pathological processes, especially in heart disease. On the basis of a gene transfer strategy, we analyzed the therapeutic efficacy of primary gene correction in a δ-sarcoglycan (δ-SG)-deficient animal model versus gene transfer of the Ca(2+) pump hSERCA2a (human sarco-endoplasmic reticulum calcium ATPase 2a), at a symptomatic stage of heart disease. Our results strongly suggest that restoration of δ-SG at this stage of disease will not lead to improved clinical outcome. However, restoration of proper Ca(2+) handling by means of amplifying SERCA2a expression in the myocardium can lead to functional improvement. Abnormalities in Ca(2+) handling play an important role in disease progression toward heart failure, and increased SERCA2a levels appear to significantly improve cardiac contraction and relaxation. Beneficial effects persist at least over a period of 6 months, and the evolution of cardiac functional parameters paralleled those of normal controls. Furthermore, we demonstrate that a plasmid formulation based on amphiphilic block copolymers can provide a safe and efficient platform for myocardial gene therapies. The use of synthetic formulations for myocardial gene transfer might thus overcome one of the major hurdles linked to viral vectors, that is, repeat administrations.
Assuntos
Terapia Genética , Insuficiência Cardíaca/terapia , Sarcoglicanas/deficiência , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica , Humanos , Masculino , Mesocricetus , Contração Miocárdica , Miocárdio/patologia , Especificidade de Órgãos , Volume Sistólico , Pressão VentricularRESUMO
Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.