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1.
J Cachexia Sarcopenia Muscle ; 10(5): 1102-1115, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31140761

RESUMO

BACKGROUND: Chronic heart failure (CHF) leads to diaphragm myopathy that significantly impairs quality of life and worsens prognosis. In this study, we aimed to assess the efficacy of a recently discovered small-molecule inhibitor of MuRF1 in treating CHF-induced diaphragm myopathy and loss of contractile function. METHODS: Myocardial infarction was induced in mice by ligation of the left anterior descending coronary artery. Sham-operated animals (sham) served as controls. One week post-left anterior descending coronary artery ligation animals were randomized into two groups-one group was fed control rodent chow, whereas the other group was fed a diet containing 0.1% of the compound ID#704946-a recently described MuRF1-interfering small molecule. Echocardiography confirmed development of CHF after 10 weeks. Functional and molecular analysis of the diaphragm was subsequently performed. RESULTS: Chronic heart failure induced diaphragm fibre atrophy and contractile dysfunction by ~20%, as well as decreased activity of enzymes involved in mitochondrial energy production (P < 0.05). Treatment with compound ID#704946 in CHF mice had beneficial effects on the diaphragm: contractile function was protected, while mitochondrial enzyme activity and up-regulation of the MuRF1 and MuRF2 was attenuated after infarct. CONCLUSIONS: Our murine CHF model presented with diaphragm fibre atrophy, impaired contractile function, and reduced mitochondrial enzyme activities. Compound ID#704946 rescued from this partially, possibly by targeting MuRF1/MuRF2. However, at this stage of our study, we refrain to claim specific mechanism(s) and targets of compound ID#704946, because the nature of changes after 12 weeks of feeding is likely to be complex and is not necessarily caused by direct mechanistic effects.


Assuntos
Diafragma/metabolismo , Diafragma/fisiopatologia , Insuficiência Cardíaca/complicações , Proteínas Musculares/antagonistas & inibidores , Proteínas com Motivo Tripartido/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Linhagem Celular , Doença Crônica , Diafragma/efeitos dos fármacos , Ecocardiografia , Feminino , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Contração Muscular/efeitos dos fármacos , Proteômica/métodos
2.
J Cachexia Sarcopenia Muscle ; 8(6): 939-953, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28887874

RESUMO

BACKGROUND: Muscle ring finger 1 (MuRF1) is a muscle-specific ubiquitin E3 ligase activated during clinical conditions associated with skeletal muscle wasting. Yet, there remains a paucity of therapeutic interventions that directly inhibit MuRF1 function, particularly in vivo. The current study, therefore, developed a novel compound targeting the central coiled coil domain of MuRF1 to inhibit muscle wasting in cardiac cachexia. METHODS: We identified small molecules that interfere with the MuRF1-titin interaction from a 130 000 compound screen based on Alpha Technology. A subset of nine prioritized compounds were synthesized and administrated during conditions of muscle wasting, that is, to C2C12 muscle cells treated with dexamethasone and to mice treated with monocrotaline to induce cardiac cachexia. RESULTS: The nine selected compounds inhibited MuRF1-titin complexation with IC50 values <25 µM, of which three were found to also inhibit MuRF1 E3 ligase activity, with one further showing low toxicity on cultured myotubes. This last compound, EMBL chemical core ID#704946, also prevented atrophy in myotubes induced by dexamethasone and attenuated fibre atrophy and contractile dysfunction in mice during cardiac cachexia. Proteomic and western blot analyses showed that stress pathways were attenuated by ID#704946 treatment, including down-regulation of MuRF1 and normalization of proteins associated with apoptosis (BAX) and protein synthesis (elF2B-delta). Furthermore, actin ubiquitinylation and proteasome activity was attenuated. CONCLUSIONS: We identified a novel compound directed to MuRF1's central myofibrillar protein recognition domain. This compound attenuated in vivo muscle wasting and contractile dysfunction in cardiac cachexia by protecting de novo protein synthesis and by down-regulating apoptosis and ubiquitin-proteasome-dependent proteolysis.


Assuntos
Caquexia/patologia , Caquexia/fisiopatologia , Coração/fisiopatologia , Proteínas Musculares/antagonistas & inibidores , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Miocárdio/patologia , Proteínas com Motivo Tripartido/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Biomarcadores , Caquexia/tratamento farmacológico , Caquexia/etiologia , Linhagem Celular , Dexametasona/farmacologia , Descoberta de Drogas , Humanos , Camundongos , Contração Muscular/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/etiologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Transdução de Sinais
3.
Biomed Res Int ; 2016: 1627184, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27812527

RESUMO

Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage.


Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/virologia , Mediadores da Inflamação/metabolismo , Parvovirus B19 Humano/genética , Animais , Proteínas do Capsídeo/imunologia , Modelos Animais de Doenças , Ecocardiografia , Epitopos/imunologia , Perfilação da Expressão Gênica , Hepatite Viral Animal/imunologia , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Transcriptoma , Vacinas
5.
Proc Natl Acad Sci U S A ; 113(3): 746-50, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26733679

RESUMO

The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic ß2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.


Assuntos
Saúde , Homeostase , Doenças do Sistema Nervoso/patologia , Junção Neuromuscular/patologia , Sistema Nervoso Simpático/patologia , Transporte Ativo do Núcleo Celular , Animais , Técnicas Biossensoriais , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Músculo Esquelético/inervação , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fenótipo , Transdução de Sinais , Simpatectomia , Sistema Nervoso Simpático/metabolismo , Fatores de Transcrição/metabolismo
6.
Autophagy ; 10(1): 123-36, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24220501

RESUMO

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63(-/-) mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Proteínas de Choque Térmico/metabolismo , Proteínas Musculares/metabolismo , Receptores Nicotínicos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Aminoácidos/deficiência , Animais , Biomarcadores/metabolismo , Endocitose , Endossomos/metabolismo , Jejum , Imunofluorescência , Marcação por Isótopo , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Denervação Muscular , Músculos/inervação , Músculos/metabolismo , Músculos/patologia , Junção Neuromuscular/metabolismo , Fagossomos/metabolismo , Estabilidade Proteica , Proteína Sequestossoma-1 , Sinapses/metabolismo , Proteínas com Motivo Tripartido , Regulação para Cima
7.
Age (Dordr) ; 35(5): 1663-74, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22956146

RESUMO

Muscle atrophy is a process of muscle wasting induced under a series of catabolic stress conditions, such as denervation, disuse, cancer cachexia, heart and renal failure, AIDS, and aging. Neuromuscular junctions (NMJs), the synapses between motor neurons and muscle fibers undergo major changes in atrophying muscles, ranging from mild morphological alterations to complete disintegration. In this study, we hypothesized that remodeling of NMJs and muscle atrophy could be linked together. To test this, we examined if a major atrophy-promoting E3 ubiquitin ligase, MuRF1, is involved in the maintenance of NMJs. Immunofluorescence revealed that MuRF1 is highly enriched close to the NMJ. Affinity precipitation and in vivo imaging showed that MuRF1 interacts in endocytic structures with both, acetylcholine receptor, the primary postsynaptic protein of the NMJ, as well as with Bif-1, an autophagy- and endocytosis-regulating factor. In vivo imaging, radio labeling, and weighing approaches demonstrated that metabolic destabilization of acetylcholine receptors and muscle atrophy induced by denervation were significantly rescued in MuRF1-KO animals. Notably, interaction with Bif-1, and the rescue of AChR lifetime and muscle atrophy were specific to MuRF1 but not MuRF2. Our data demonstrate an involvement of MuRF1 in membrane protein-turnover, including the degradation of AChRs at the NMJ under atrophying conditions where MuRF1 also interacts and associates with Bif-1.


Assuntos
Lisossomos/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Receptores Nicotínicos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Modelos Animais de Doenças , Endocitose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Junção Neuromuscular/metabolismo , Proteínas com Motivo Tripartido
8.
J Biomed Biotechnol ; 2010: 693741, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20625437

RESUMO

MuRF1 is a member of the TRIM/RBCC superfamily, a gene family that encompasses a large variety of proteins, all sharing the conserved TRIM (Tripartite Motive) sequential array of RING, B-box, and coiled-coil domains. Within this family, MuRF1(also named TRIM63) is a specialized member that contributes to the development of muscle atrophy and sarcopenia. Here we studied MuRF1's role in muscle atrophy during muscle unloading induced by hindlimb suspension. Consistent with previous studies, we found that MuRF1 inactivation leads to an attenuated muscle atrophy response. The amount of protection was higher as compared to the denervation model, and within the 10 day-suspension period the soleus muscle was spared from atrophy in MuRF1-KO mice. Contractility studies on hindlimb suspended muscle tissues suggested that MuRF1's functions extend beyond muscle trophicity and implicate MuRF1 in muscle fatigue and MLC phosphorylation control: soleus muscle from MuRF1-KO mice fatigued significantly faster and in addition showed a reduced posttetanic twitch potentiation. Thus the present work further established the role of MuRF1 in muscle atrophy and for the first time shows that MuRF1 plays a role in muscle fatigue and twitch potentiation.


Assuntos
Elevação dos Membros Posteriores , Fadiga Muscular/fisiologia , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Cadeias Leves de Miosina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/deficiência , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Tamanho do Órgão , Fosforilação , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/deficiência
9.
J Mol Biol ; 379(4): 666-77, 2008 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-18468620

RESUMO

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase beta (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1's role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.


Assuntos
Metabolismo dos Carboidratos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Insulina/metabolismo , Secreção de Insulina , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Proteoma , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Distribuição Tecidual , Proteínas com Motivo Tripartido
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