RESUMO
Skeletal muscle is a highly plastic tissue. The role of heat shock protein 72 (Hsp72) in heat stress-induced skeletal muscle hypertrophy has been well demonstrated; however, the precise mechanisms remain unclear. Essential amino acids, such as leucine, mainly mediate muscle protein synthesis. We investigated the effects of pre-heating and increased Hsp72 expression on the mechanistic target of rapamycin (mTOR) signaling and protein synthesis following leucine administration in rat gastrocnemius muscle. To ensure increased Hsp72 expression in both the red and white portions of the muscle, one leg of male Wistar rats (10-week-old, n = 23) was heat-stressed in 43 °C water for 30 min twice at a 48-h-interval (heat-stressed leg, HS leg). The contralateral leg served as a non-heated internal control (CT leg). After the recovery period (48 h), rats were divided into the pre-administration or oral leucine administration groups. We harvested the gastrocnemius muscle (red and white parts) prior to administration and 30 and 90 min after leucine treatment (n = 7-8 per group) and intramuscular signaling responses to leucine ingestion were determined using western blotting. Heat stress significantly upregulated the expression of Hsp72 and was not altered by leucine administration. Although the phosphorylation levels of mTOR/S6K1 and ERK were similar regardless of heating, 4E-BP1 was less phosphorylated in the HS legs than the CT legs after leucine administration in the red portion of the muscles (P < 0.05). Moreover, c-Myc expression differed significantly after leucine administration in both the red and white portions of the muscles. Our findings indicate that following oral leucine administration, pre-heating partially blunted the muscle protein synthesis signaling response in the rat gastrocnemius muscle.
Assuntos
Calefação , Transdução de Sinais , Ratos , Masculino , Animais , Leucina/farmacologia , Ratos Sprague-Dawley , Ratos Wistar , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologia , Músculo Esquelético/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacologia , Suplementos NutricionaisRESUMO
BACKGROUND: Although chronic treatment with glucocorticoids, such as dexamethasone, is frequently associated with muscle atrophy, effective and safe therapeutics for treating muscle atrophy remain elusive. Jakyak-gamcho-tang (JGT), a decoction of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma, has long been used to relieve muscle tension and control muscle cramp-related pain. However, the effects of JGT on glucocorticoid-induced muscle atrophy are yet to be comprehensively clarified. PURPOSE: The objective of the current study was to validate the protective effect of JGT in dexamethasone-induced muscle atrophy models and elucidate its underlying mechanism through integrated in silico - in vitro - in vivo studies. STUDY DESIGN AND METHODS: Differential gene expression was preliminarily analyzed using the RNA-seq data to determine the effects of JGT on C2C12 myotubes. The protective effects of JGT were further validated in dexamethasone-treated C2C12 myotubes by assessing cell viability, myotube integrity, and mitochondrial function or in C57BL/6 N male mice with dexamethasone-induced muscle atrophy by evaluating muscle mass and physical performance. Transcriptomic pathway analysis was also performed to elucidate the underlying mechanism. RESULTS: Based on preliminary gene set enrichment analysis using the RNA-seq data, JGT regulated various pathways related to muscle differentiation and regeneration. Dexamethasone-treated C2C12 myotubes and muscle tissues of atrophic mice displayed substantial muscle protein degradation and muscle loss, respectively, which was efficiently alleviated by JGT treatment. Importantly, JGT-mediated protective effects were associated with observations such as preservation of mitochondrial function, upregulation of myogenic signaling pathways, including protein kinase B/mammalian target of rapamycin/forkhead box O3, inhibition of ubiquitin-mediated muscle protein breakdown, and downregulation of inflammatory and apoptotic pathways induced by dexamethasone. CONCLUSION: To the best of our knowledge, this is the first report to demonstrate that JGT could be a potential pharmaceutical candidate to prevent muscle atrophy induced by chronic glucocorticoid treatment, highlighting its known effects for relieving muscle spasms and pain. Moreover, transcriptomic pathway analysis can be employed as an efficient in silico tool to predict novel pharmacological candidates and elucidate molecular mechanisms underlying the effects of herbal medications comprising diverse biologically active ingredients.
Assuntos
Medicamentos de Ervas Chinesas , Glucocorticoides , Glycyrrhiza , Paeonia , Masculino , Camundongos , Animais , Camundongos Endogâmicos C57BL , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/tratamento farmacológico , Fibras Musculares Esqueléticas , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacologia , Proteínas Musculares/uso terapêutico , Dexametasona/farmacologia , Dor , MamíferosRESUMO
Background: Advanced glycation end products (AGEs) impair vascular physiology in Diabetes mellitus (DM). However, the underlying mechanisms remain unclear. Vascular large conductance calcium-activated potassium (BK) channels play important roles in coronary arterial function.Purpose: Our study aimed to investigate the regulatory role of AGEs in BK channels.Research Design: Using gavage of vehicle (V, normal saline) or aminoguanidine (A) for 8 weeks, normal and diabetic rats were divided into four groups: C+V group, DM+V group, C+A group, and DM+A group.Study Sample: Coronary arteries from different groups of rats and human coronary smooth muscle cells were used in this study.Data Collection and Analysis: Data were presented as mean ± SEM (standard error of mean). Student's t-test was used to compare data between two groups. One-way ANOVA with post-hoc LSD analysis was used to compare data between multiple groups.Results: Compared to the C+V group, vascular contraction induced by iberiotoxin (IBTX), a BK channel inhibitor, was impaired, and BK channel densities decreased in the DM+V group. However, aminoguanidine administration reduced the impairment. Protein expression of BK-ß1, phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein kinase B (PKB or Akt) were down-regulated, while F-box protein 32 (FBXO32) expression increased in the DM+V group and in high glucose (HG) cultured human coronary smooth muscle cells. Treatment with aminoguanidine in vitro and in vivo could reverse the above protein expression. The effect of aminoguanidine on the improvement of BK channel function by inhibiting the generation of AGEs was reversed by adding MK2206 (Akt inhibitor) or Compound C (AMPK inhibitor) in HG conditions in vitro.Conclusions: AGEs aggravate BK channel dysfunction via the AMPK/Akt/FBXO32 signaling pathway.
Assuntos
Vasos Coronários , Diabetes Mellitus Experimental , Ratos , Humanos , Animais , Vasos Coronários/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Transdução de Sinais , Produtos Finais de Glicação Avançada/metabolismo , Miócitos de Músculo Liso , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/farmacologiaRESUMO
BACKGROUND: Cysteine-rich protein 2 (CSRP2) is discovered as oncogene. The study aims to investigate the clinical significance and potential mechanism of CSRP2 in head and neck squamous cell carcinoma (HNSCC). METHODS: CSRP2 expression was explored by immunohistochemistry tissue microarrays and Western blotting in HNSCC. The effect of CSRP2 on the cancer stemness and epithelial-to-mesenchymal transition (EMT) of HNSCC cells was investigated by sphere formation, wound healing, and transwell assays. The vitro and vivo experiments revealed that CSRP2 modulated cancer stemness and EMT phenotypes in HNSCC. RESULTS: CSRP2 was overexpressed in HNSCC patients and presented poor prognosis. CSRP2 knockdown inhibited the migration and invasion ability of the HNSCC cells. And CSRP2 expression was closely associated with CSCs markers, EMT-transcription factor, new oncoprotein, and immune checkpoint. CONCLUSION: The overexpression of CSRP2 indicates poor prognosis and plays a key role in maintaining the cancer cell stemness and EMT.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/genética , Fatores de Transcrição/genética , Fenótipo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Movimento Celular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacologia , Proteínas Nucleares/genética , Proteínas com Domínio LIM/genéticaRESUMO
Alcoholic liver cirrhosis (ALC) is accompanied by sarcopenia. The aim of this study was to investigate the acute effects of balanced parenteral nutrition (PN) on skeletal muscle protein turnover in ALC. Eight male patients with ALC and seven age- and sex-matched healthy controls were studied for 3 h of fasting followed by 3 h of intravenous PN (SmofKabiven 1,206 mL: amino acid = 38 g, carbohydrates = 85 g, and fat = 34 g) 4 mL/kg/h. We measured leg blood flow and sampled paired femoral arteriovenous concentrations and quadriceps muscle biopsies while providing a primed continuous infusion of [ring-2d5]-phenylalanine to quantify muscle protein synthesis and breakdown. Patients with ALC exhibited shorter 6-min walking distance (ALC: 487 ± 38 vs. controls: 722 ± 14 m, P < 0.05), lower hand-grip strength (ALC: 34 ± 2 vs. controls: 52 ± 2 kg, P < 0.05), and computed tomography (CT)-verified leg muscle loss (ALC: 5,922 ± 246 vs. controls: 8,110 ± 345 mm2, P < 0.05). Net leg muscle phenylalanine uptake changed from negative (muscle loss) during fasting to positive (muscle gain) in response to PN (ALC: -0.18 ± +0.01 vs. 0.24 ± 0.03 µmol/kg muscle·min-1; P < 0.001 and controls: -0.15 ± 0.01 vs. 0.09 ± 0.01 µmol/kg muscle·min-1; P < 0.001) but with higher net muscle phenylalanine uptake in ALC than controls (P < 0.001). Insulin concentrations were substantially higher in patients with ALC during PN. Our results suggest a higher net muscle phenylalanine uptake during a single infusion of PN in stable patients with ALC with sarcopenia compared with healthy controls.NEW & NOTEWORTHY Muscle protein turnover responses to parenteral nutritional (PN) supplementation have not previously been studied in stable alcoholic liver cirrhosis (ALC). We applied stable isotope tracers of amino acids to directly quantify net muscle protein turnover responses to PN in sarcopenic males with ALC and healthy controls. We found a higher net muscle protein gain in ALC during PN, thereby providing the physiological rationale for future clinical trials of PN as a potential countermeasure to sarcopenia.
Assuntos
Músculo Esquelético , Nutrição Parenteral , Sarcopenia , Humanos , Masculino , Aminoácidos/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática Alcoólica/terapia , Cirrose Hepática Alcoólica/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacologia , Músculo Esquelético/metabolismo , Fenilalanina , Sarcopenia/complicações , Estudos de Casos e ControlesRESUMO
Age-related loss of muscle mass, strength, and performance, commonly referred to as sarcopenia, has wide-ranging detrimental effects on human health, the ramifications of which can have serious implications for both morbidity and mortality. Various interventional strategies have been proposed to counteract sarcopenia, with a particular emphasis on those employing a combination of exercise and nutrition. However, the efficacy of these interventions can be confounded by an age-related blunting of the muscle protein synthesis response to a given dose of protein/amino acids, which has been termed "anabolic resistance." While the pathophysiology of sarcopenia is undoubtedly complex, anabolic resistance is implicated in the progression of age-related muscle loss and its underlying complications. Several mechanisms have been proposed as underlying age-related impairments in the anabolic response to protein consumption. These include decreased anabolic molecular signaling activity, reduced insulin-mediated capillary recruitment (thus, reduced amino acid delivery), and increased splanchnic retention of amino acids (thus, reduced availability for muscular uptake). Obesity and sedentarism can exacerbate, or at least facilitate, anabolic resistance, mediated in part by insulin resistance and systemic inflammation. This narrative review addresses the key factors and contextual elements involved in reduction of the acute muscle protein synthesis response associated with aging and its varied consequences. Practical interventions focused on dietary protein manipulation are proposed to prevent the onset of anabolic resistance and mitigate its progression.
Assuntos
Sarcopenia , Humanos , Sarcopenia/prevenção & controle , Músculo Esquelético/metabolismo , Envelhecimento/fisiologia , Aminoácidos , Proteínas Musculares/metabolismo , Proteínas Musculares/farmacologia , Força MuscularRESUMO
Proteasomal activity is compromised in diabetic hearts that contributes to proteotoxic stresses and cardiac dysfunction. Osteocrin (OSTN) acts as a novel exercise-responsive myokine and is implicated in various cardiac diseases. Herein, we aim to investigate the role and underlying molecular basis of OSTN in diabetic cardiomyopathy (DCM). Mice received a single intravenous injection of the cardiotrophic adeno-associated virus serotype 9 to overexpress OSTN in the heart and then were exposed to intraperitoneal injections of streptozotocin (STZ, 50 mg/kg) for consecutive 5 days to generate diabetic models. Neonatal rat cardiomyocytes were isolated and stimulated with high glucose to verify the role of OSTN in vitro. OSTN expression was reduced by protein kinase B/forkhead box O1 dephosphorylation in diabetic hearts, while its overexpression significantly attenuated cardiac injury and dysfunction in mice with STZ treatment. Besides, OSTN incubation prevented, whereas OSTN silence aggravated cardiomyocyte apoptosis and injury upon hyperglycemic stimulation in vitro. Mechanistically, OSTN treatment restored protein kinase G (PKG)-dependent proteasomal function, and PKG or proteasome inhibition abrogated the protective effects of OSTN in vivo and in vitro. Furthermore, OSTN replenishment was sufficient to prevent the progression of pre-established DCM and had synergistic cardioprotection with sildenafil. OSTN protects against DCM via restoring PKG-dependent proteasomal activity and it is a promising therapeutic target to treat DCM.
Assuntos
Apoptose/efeitos dos fármacos , Cardiomiopatias Diabéticas/prevenção & controle , Proteínas Musculares/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/farmacologia , Animais , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/patologia , Modelos Animais de Doenças , Proteína Forkhead Box O1/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosforilação , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling.
Assuntos
Receptores do Fator Natriurético Atrial/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células HEK293 , Células HeLa , Humanos , Ligantes , Proteínas Musculares/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de Transcrição/farmacologiaRESUMO
The poor gel strength and microbial infection of conventional chicken myofibrillar protein (CMP) gels have severely limited the application. Here, plasma activated water (PAW) instead of normal water was used to prepare CMP gels. PAW prepared by treating deionized water with plasma jet was incubated with CMPs and followed by heating to prepare CMP gels. Effects of PAW on CMP gels were assessed in terms of basic physicochemical properties, network structure, and antibacterial activity. The results showed that PAW treatment accelerated the aggregation of CMPs and increased the strength and water holding capacity of CMP gels. Due to the presence of NO and NO2 free radicals in PAW, the prepared CMP gels were endowed with antibacterial activity against Salmonella Enteritidis and Staphylococcus aureus. The new method of PAW-induced CMP gels will have the prospect of improving the quality of gels and extending the shelf life of chicken gel products.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Galinhas , Proteínas Musculares/química , Proteínas Musculares/farmacologia , Gases em Plasma/química , Água/química , Animais , GéisRESUMO
Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1â h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.
Assuntos
Proteínas de Ligação ao Cálcio/farmacologia , Proteínas Musculares/farmacologia , Proteolipídeos/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , DNA Complementar/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Oxirredução/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , TemperaturaRESUMO
BACKGROUND: Marine fish meat has been widely used for the extraction of bioactive peptides. This study was aimed to optimize the preparation of monkfish muscle peptides (LPs) using response surface methodology (RSM) and explore the antioxidant activities of <1 kDa LPs. METHODS: Peptides were prepared from the muscles of monkfish (Lophius litulon), and five proteases were tested to hydrolyze muscle proteins. The hydrolysate that was treated using neutrase showed the highest degree of hydrolysis (DH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities. RESULTS: The optimized conditions were as follows: water/material ratio of 5.4:1, a time span of 5 h, pH of 7.0, enzyme concentration of 2000 U/g, and temperature of 45 °C; the maximum DPPH scavenging activity and DH were 92.861% and 19.302%, respectively. LPs exhibited appreciable antioxidant activities, including DPPH radical, hydroxyl radical, 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) radical, and superoxide anion scavenging activities. LPs attenuated H2O2-related oxidative injury in RAW264.7 cells, reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) levels. CONCLUSION: We concluded that LPs could be an ideal source of bioactive peptides from monkfish and also have pharmaceutical potential.
Assuntos
Antioxidantes/farmacologia , Proteínas de Peixes/farmacologia , Peróxido de Hidrogênio/toxicidade , Macrófagos/efeitos dos fármacos , Proteínas Musculares/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Catalase/metabolismo , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/metabolismo , Glutationa Peroxidase/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Malondialdeído/metabolismo , Camundongos , Proteínas Musculares/isolamento & purificação , Proteínas Musculares/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Células RAW 264.7 , Superóxido Dismutase/metabolismoRESUMO
Fibrosis is a key pathological event during muscle aging that accelerates the development of sarcopenia. We show that sarcolipin (SLN) is highly expressed during aging, promotes intracellular calcium overload and participates in impaired myogenic differentiation. d-Galactose (D-gal) was used to induce senescence in C2C12 myoblasts. Conventional AAV-mediated SLN knockdown cells were used to study the role of SLN in muscle physiology and pathophysiology. C2C12 cells were treated with D-gal, which promoted fibrosis and SLN upregulation. The expression of TGF-ß1 and α-SMA, which participate in myogenic transdifferentiation, were also elevated. C2C12 cells with reduced sarcolipin expression produced decreased amounts of collagen. Our study identified an unrecognized role of SLN in regulating myogenic transdifferentiation during aging-associated skeletal muscle cell fibrosis. Targeting SLN may be a novel therapeutic strategy to relieve sarcopenia-associated muscle fibrosis.
Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Proteínas Musculares/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Proteolipídeos/farmacologia , Sarcopenia/patologia , Animais , Cálcio/metabolismo , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Fibrose , Desenvolvimento Muscular/efeitos dos fármacos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Sarcopenia/complicações , Sarcopenia/metabolismoRESUMO
Nebulin, encoded by NEB, is a giant skeletal muscle protein of about 6669 amino acids which forms an integral part of the sarcomeric thin filament. In recent years, the nebula around this protein has been largely lifted resulting in the discovery that nebulin is critical for a number of tasks in skeletal muscle. In this review, we firstly discussed nebulin's role as a structural component of the thin filament and the Z-disk, regulating the length and the mechanical properties of the thin filament as well as providing stability to myofibrils by interacting with structural proteins within the Z-disk. Secondly, we reviewed nebulin's involvement in the regulation of muscle contraction, cross-bridge cycling kinetics, Ca2+-homeostasis and excitation contraction (EC) coupling. While its role in Ca2+-homeostasis and EC coupling is still poorly understood, a large number of studies have helped to improve our knowledge on how nebulin affects skeletal muscle contractile mechanics. These studies suggest that nebulin affects the number of force generating actin-myosin cross-bridges and may also affect the force that each cross-bridge produces. It may exert this effect by interacting directly with actin and myosin and/or indirectly by potentially changing the localisation and function of the regulatory complex (troponin and tropomyosin). Besides unravelling the biology of nebulin, these studies are particularly helpful in understanding the patho-mechanism of myopathies caused by NEB mutations, providing knowledge which constitutes the critical first step towards the development of therapeutic interventions. Currently, effective treatments are not available, although a number of therapeutic strategies are being investigated.
Assuntos
Proteínas Musculares/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Humanos , Proteínas Musculares/farmacologiaRESUMO
Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC.
Assuntos
Doenças do Gato/tratamento farmacológico , Proteínas Mitocondriais/farmacologia , Transportadores de Ácidos Monocarboxílicos/farmacologia , Neoplasias Bucais/veterinária , Carcinoma de Células Escamosas de Cabeça e Pescoço/veterinária , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/farmacologia , Animais , Gatos , Linhagem Celular Tumoral , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Transportadores de Ácidos Monocarboxílicos/genética , Neoplasias Bucais/tratamento farmacológico , Proteínas Musculares/genética , Proteínas Musculares/farmacologia , Análise de Sequência de RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológicoRESUMO
BACKGROUND: Myopalladin (MYPN) is a striated muscle-specific, immunoglobulin-containing protein located in the Z-line and I-band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss-of-function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. METHODS: Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. RESULTS: MKO mice were 13% smaller compared with wild-type controls and exhibited a 48% reduction in myofibre cross-sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild-type mice, MKO mice showed progressively decreased exercise capability, Z-line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z-line widening starting from 8 months of age. RNA-sequencing analysis revealed down-regulation of serum response factor (SRF)-target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF-cofactor myocardin-related transcription factor A (MRTF-A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF-A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF-A-mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. CONCLUSIONS: Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z-line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
Assuntos
Proteínas Musculares/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Fator de Resposta Sérica/efeitos dos fármacos , Animais , Feminino , Humanos , Camundongos , Camundongos Knockout , Proteínas Musculares/farmacologiaRESUMO
MDR and tumor migration and invasion are still the main obstacles to effective breast cancer chemotherapies. Transgelin 2 has recently been shown to induce drug resistance, tumor migration, and invasion. The aim of this study was to determine the biological functions of Transgelin 2 and the mechanism underlying how Transgelin 2 induces paclitaxel (PTX) resistance and the migration and invasion of breast cancer. We detected that the protein level of Transgelin 2 was significantly upregulated in breast cancer tissues compared with adjacent nontumor tissues. A bioinformatics analysis indicated that Transgelin 2 was significantly related to clinicopathologic parameters and patient prognosis. Overexpression of Transgelin 2 enhanced the migration and invasion of human breast cancer cells and decreased the sensitivity of breast cancer cells to paclitaxel. Meanwhile, the tumorigenesis and metastasis of breast cancer cells were also enhanced by Transgelin 2 overexpression in vivo Moreover, Transgelin 2 overexpression activated the PI3K/Akt/GSK-3ß pathway by increasing the phosphorylation levels of Akt and GSK-3ß and decreasing the expression of PTEN. We also found that Transgelin 2 could directly interact with PTEN and was located upstream of PTEN. Furthermore, the PI3K/Akt pathway inhibitor MK-2206 reversed the resistance to paclitaxel and inhibited the migration and invasion of breast cancer cells. These findings indicate that Transgelin 2 promotes paclitaxel resistance and the migration and invasion of breast cancer by directly interacting with PTEN and activating the PI3K/Akt/GSK-3ß pathway. Transgelin 2 may therefore be useful as a novel biomarker and therapeutic target for breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas dos Microfilamentos/uso terapêutico , Proteínas Musculares/uso terapêutico , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos/farmacologia , Proteínas Musculares/farmacologiaRESUMO
KEY POINTS: The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied. Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform. Recovery from desensitization is determined by the AChR isoform-specific cytoplasmic M3-M4 domain. The co-expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering. These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions. ABSTRACT: The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle-type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole-cell currents in muscle-type (TE671/CN21) and non-muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P < 0.0001). Chimeric experiments showed that recovery from desensitization was determined by the M3-M4 cytoplasmic loops of the γ- and ε-subunits. Expression of rapsyn in TE671/CN21 cells induced AChR aggregation and also, surprisingly, shortened recovery time constants in both fetal and adult AChRs. However, this was not dependent on clustering because rapsyn also facilitated recovery from desensitization in HEK293 cells expressing a δ-R375H AChR mutant that did not form clusters in C2C12 myotubes. Thus, rapsyn interactions with AChRs lead not only to clustering, but also to a clustering independent faster recovery from desensitization. Both effects of rapsyn could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth.
Assuntos
Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/farmacologia , Receptores Nicotínicos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismoRESUMO
RATIONALE: Physical exercise provides benefits for various organ systems, and some of systemic effects of exercise are mediated through modulation of muscle-derived secreted factors, also known as myokines. Myonectin/C1q (complement component 1q)/TNF (tumor necrosis factor)-related protein 15/erythroferrone is a myokine that is upregulated in skeletal muscle and blood by exercise. OBJECTIVE: We investigated the role of myonectin in myocardial ischemic injury. METHODS AND RESULTS: Ischemia-reperfusion in myonectin-knockout mice led to enhancement of myocardial infarct size, cardiac dysfunction, apoptosis, and proinflammatory gene expression compared with wild-type mice. Conversely, transgenic overexpression of myonectin in skeletal muscle reduced myocardial damage after ischemia-reperfusion. Treadmill exercise increased circulating myonectin levels in wild-type mice, and it reduced infarct size after ischemia-reperfusion in wild-type mice, but not in myonectin-knockout mice. Treatment of cultured cardiomyocytes with myonectin protein attenuated hypoxia/reoxygenation-induced apoptosis via S1P (sphingosine-1-phosphate)-dependent activation of cAMP/Akt cascades. Similarly, myonectin suppressed inflammatory response to lipopolysaccharide in cultured macrophages through the S1P/cAMP/Akt-dependent signaling pathway. Moreover, blockade of S1P-dependent pathway reversed myonectin-mediated reduction of myocardial infarct size in mice after ischemia-reperfusion. CONCLUSIONS: These data indicate that myonectin functions as an endurance exercise-induced myokine which ameliorates acute myocardial ischemic injury by suppressing apoptosis and inflammation in the heart, suggesting that myonectin mediates some of the beneficial actions of exercise on cardiovascular health.
Assuntos
Citocinas/metabolismo , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Condicionamento Físico Animal/métodos , Animais , Apoptose , Células Cultivadas , AMP Cíclico/metabolismo , Citocinas/genética , Citocinas/farmacologia , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/farmacologia , Músculo Esquelético/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Dietary proteins harbour bioactive peptides that exert various physiological activities. Chicken meat prepared from spent layers from the egg industry is an inexpensive source of protein for the production of bioactive peptides. This study explored the effect of hen muscle-derived peptides prepared by enzymatic hydrolysis on immune functions. The hydrolysate was incorporated into the diet of weanling Sprague-Dawley rats (n = 8 per diet) for 3 weeks at 2% or 5% addition (w/w diet). At a dose of 5% (w/w) the hydrolysate exhibited immunomodulatory effects on splenocytes, including a lower proportion of OX6+ (professional antigen presenting cells) and a higher proportion of CD11b/c+ cells (macrophages/monocytes) (p < 0.05) compared to the isonitrogenous control diet. Meanwhile, the production of anti-inflammatory cytokine interleukin (IL)-10 by splenocytes stimulated ex vivo with mitogens was significantly higher from hydrolysate treatment; there was no significant difference in the other cytokines (IL-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6 and IL-2) investigated. Supplementing with the hydrolysate did not alter the growth, food intake and organ weights in young rodents. These results indicated that the spent hen muscle protein hydrolysate has the potential to be developed for value-added products with anti-inflammatory properties.
Assuntos
Anti-Inflamatórios/química , Interleucina-10/metabolismo , Proteínas Musculares/química , Hidrolisados de Proteína/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/metabolismo , Galinhas , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-2/genética , Interleucina-2/metabolismo , Masculino , Proteínas Musculares/farmacologia , Hidrolisados de Proteína/química , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Excessive concentrations of angiotensin-converting enzyme (ACE) can give rise to high blood pressure, and is harmful to the body. ACE inhibitory peptides from food proteins are considered good sources of function food. However, the preparation of ACE inhibitory peptides by classical method faces many challenges. Three novel ACE inhibitory peptides were identified by in silico methods, and showed potent activity against ACE in vitro. The simulation hydrolysis of nebulin was performed with ExPASy PeptideCutter program. Potential activity, solubility, and absorption, distribution, metabolism, excretion, and toxicity properties of generated peptides were predicted using program online. Molecular docking displayed that EGF, HGR, and VDF were docked into the S1 and S2 pockets of ACE. Meanwhile, Phe and Arg at the C-terminal enhance ACE affinity. The IC50 values of EGF, HGR, and VDF were 474.65 ± 0.08, 106.21 ± 0.52, and 439.27 ± 0.09 µM, respectively. Three peptides EGF, HGR, and VDF from Oncorhynchus mykiss nebulin were identified, and the molecular mechanism between ACE and peptides was clarified using in silico methods. The results suggested that Oncorhynchus mykiss nebulin would be an attractive raw material of antihypertensive nutraceutical ingredients. PRACTICAL APPLICATION: This study has shown the potential of Oncorhynchus mykiss nebulin as good sources for producing ACE inhibitory peptides. According to this finding, in silico approach is the feasible way for prediction and identification of food-derived ACE inhibitory peptides in emerging nutraceutical field.