RESUMO
BACKGROUND: Dietary fish oil (DFO) has been identified as a micronutrient supplement with the potential to improve musculoskeletal health in old age. Few data are available for effects of DFO on muscle contractility, despite the significant negative impact of muscle weakness on age-related health outcomes. Accordingly, the effects of a DFO intervention on the contractile function and proteomic profile of adult and aged in an animal model of aging were investigated. METHODS: This preliminary study evaluated 14 adult (8 months) and 12 aged (22 months) male, Sprague-Dawley rats consuming a DFO-supplemented diet or a control diet for 8 weeks (7 adult and 6 aged/dietary group). Animal weight, food intake and grip strength were assessed at the start and end of the FO intervention. In situ force and contractile properties were measured in the medial gastrocnemius muscle following the intervention and muscles were processed for 2-D gel electrophoresis and proteomic analysis via liquid chromatography with tandem mass spectrometry, confirmed by immunoblotting. Effects of age, diet and age x diet interaction were evaluated by 2-way ANOVA. RESULTS: A significant (P = 0.022) main effect for DFO to increase (~ 15%) muscle contractile force was observed, without changes in muscle mass. Proteomic analysis revealed a small number of proteins that differed across age and dietary groups at least 2-fold, most of which related to metabolism and oxidative stress. In seven of these proteins (creatine kinase, triosephosphate isomerase, pyruvate kinase, parvalbumin, beta-enolase, NADH dehydrogenase and Parkin7/DJ1), immunoblotting corroborated these findings. Parvalbumin showed only an effect of diet (increased with DFO) (P = 0.003). Significant age x diet interactions were observed in the other proteins, generally demonstrating increased expression in adult and decreased expression aged rats consuming DFO (all P > 0.011). However, correlational analyses revealed no significant associations between contractile parameters and protein abundances. CONCLUSIONS: Results of this preliminary study support the hypothesis that DFO can enhance musculoskeletal health in adult and aged muscles, given the observed improvement in contractile function. The fish oil supplement also alters protein expression in an age-specific manner, but the relationship between proteomic and contractile responses remains unclear. Further investigation to better understand the magnitude and mechanisms muscular effects of DFO in aged populations is warranted.
Assuntos
Óleos de Peixe/farmacologia , Contração Muscular/efeitos dos fármacos , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Suplementos Nutricionais , Ingestão de Alimentos/efeitos dos fármacos , Proteínas de Peixes/metabolismo , Masculino , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Ratos Sprague-DawleyRESUMO
AIM: Contusion injury in aging muscle has not been studied in detail, but older adults are at risk for such injuries due to increased risk of falls. As falls in older populations are unlikely to be eliminated, interventions to minimize the negative impact of falls, including contusion injury should be pursued. Dietary fish oil (FO) is a common often supplement in older adults, which is associated with factors that might reduce or worsen the negative impact of contusion. METHODS: Here, we investigate whether 8â¯weeks of FO can blunt the impact of contusion injury in adult (nâ¯=â¯14) and aged (nâ¯=â¯12) rats. We assessed contractility and several biochemical markers in adult and aged gastrocnemius muscles 48â¯h post-contusion injury, using the uninjured muscles as controls. RESULTS: Injury reduced force production ~40% (Pâ¯<â¯0.001), sarcoplasmic reticulum calcium release by ~20% (Pâ¯=â¯0.003) and significantly increased several markers of muscle damage (i.e., protein carbonyls, Grp78 abundance (Pâ¯=â¯0.022, 0.006, respectively)), and these injury-related changes were not affected by aging. The effects of FO were limited. A main effect (Pâ¯=â¯0.018) for FO to increase the myogenic factor Myf5 was observed. In addition FO reduced the injury-associated decline in the mitophagy factor DRP1 (Pâ¯=â¯0.027). CONCLUSION: Although age-related differences in certain protein markers differed, aged muscles exhibited no greater acute functional deficits following injury. Similarly, while FO did not reduce functional deficits, it did not worsen them. However, changes in Myf5 and DRP1 with dietary FO suggest the potential to improve recovery from contusion injury, which should be investigated in future studies.
Assuntos
Envelhecimento , Contusões/terapia , Suplementos Nutricionais , Óleos de Peixe/uso terapêutico , Animais , Masculino , Contração Muscular , Ratos , Ratos Sprague-Dawley , Retículo Sarcoplasmático/metabolismoRESUMO
This study evaluated the effects of dietary ß-hydroxy-ß-methylbutyrate (HMB) combined with ß-alanine (ß-Ala) in sedentary, aged male rats. It has been suggested that dietary HMB or ß-Ala supplementation may mitigate age-related declines in muscle strength and fatigue resistance. A total of 20 aged Sprague-Dawley rats were studied. At age 20 months, 10 rats were administered a control, purified diet and 10 rats were administered a purified diet supplemented with both HMB and ß-Ala (HMB+ß-Ala) for 8 weeks (approximately equivalent to 3 and 2.4 g per day human dose). We measured medial gastrocnemius (MG) size, force, fatigability, and myosin composition. We also evaluated an array of protein markers related to muscle mitochondria, protein synthesis and breakdown, and autophagy. HMB+ß-Ala had no significant effects on body weight, MG mass, force or fatigability, myosin composition, or muscle quality. Compared with control rats, those fed HMB+ß-Ala exhibited a reduced (41%, P = 0.039) expression of muscle RING-finger protein 1 (MURF1), a common marker of protein degradation. Muscle from rats fed HMB+ß-Ala also exhibited a 45% reduction (P = 0.023) in p70s6K phosphorylation following fatiguing stimulation. These data suggest that HMB+ß-Ala at the dose studied may reduce muscle protein breakdown by reducing MURF1 expression, but has minimal effects on muscle function in this model of uncomplicated aging. They do not, however, rule out potential benefits of HMB+ß-Ala co-supplementation at other doses or durations of supplementation in combination with exercise or in situations where extreme muscle protein breakdown and loss of mass occur (e.g., bedrest, cachexia, failure-to-thrive).
Assuntos
Envelhecimento , Suplementos Nutricionais , Contração Muscular/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Sarcopenia/prevenção & controle , Comportamento Sedentário , Valeratos/farmacologia , beta-Alanina/farmacologia , Fatores Etários , Animais , Autofagia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Masculino , Fadiga Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Fosforilação , Proteólise , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sarcopenia/etiologia , Sarcopenia/metabolismo , Sarcopenia/patologia , Sarcopenia/fisiopatologia , Miosinas de Músculo Esquelético/metabolismo , Fatores de Tempo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismoRESUMO
OBJECTIVE: Calcineurin (Cn) and the nuclear factor of activated T cells (NFAT) family of transcription factors are critical in vascular smooth muscle cell (SMC) development and pathology. Here, we used a genomics approach to identify and validate NFAT gene targets activated during platelet-derived growth factor-BB (PDGF-BB)-induced SMC phenotypic modulation. METHODS AND RESULTS: Genome-wide expression arrays were used to identify genes both (1) differentially activated in response to PDGF-BB and (2) whose differential expression was reduced by both the Cn inhibitor cyclosporin A and the NFAT inhibitor A-285222. The 20 most pharmacologically sensitive genes were validated by quantitative reverse transcription-polymerase chain reaction analysis of PDGF-BB-stimulated SMCs in the presence of Cn/NFAT inhibitors, including the VIVIT peptide. In all experiments, protein C receptor (PROCR) gene activation was reduced. We showed that PROCR expression was virtually absent in untreated, quiescent SMCs. PDGF-BB stimulation, however, induced significant PROCR promoter activation and downstream protein expression in a Cn/NFAT-dependent manner. Mutation of a species-conserved, NFAT binding motif significantly attenuated PDGF-BB-induced PROCR promoter activity, thereby distinguishing NFAT as the first PROCR transcriptional activator to date. Moreover, SMC PROCR expression was upregulated in the neointima as early as 7 days following acute vascular injury in rat carotid arteries. CONCLUSION: We hereby report PROCR as a novel, NFAT-dependent gene that may be implicated in vascular restenosis and consequent inward remodeling.
Assuntos
Fatores de Coagulação Sanguínea/genética , Calcineurina/genética , Genoma/genética , Músculo Liso Vascular/patologia , Fatores de Transcrição NFATC/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Receptores de Superfície Celular/genética , Animais , Sequência de Bases , Becaplermina , Fatores de Coagulação Sanguínea/metabolismo , Calcineurina/metabolismo , Inibidores de Calcineurina , Lesões das Artérias Carótidas/metabolismo , Cateterismo/efeitos adversos , Células Cultivadas , Ciclosporina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Modelos Animais , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Pirazóis/farmacologia , Ratos , Receptores de Superfície Celular/metabolismoRESUMO
Cyclosporine A (CSA, calcineurin inhibitor) has been shown to block both vascular smooth muscle cell (VSMC) proliferation in cell culture and vessel neointimal formation following injury in vivo. The purpose of this study was to determine molecular and pathological effects of CSA on VSMCs. Using real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunofluorescence microscopy, we show that CSA up-regulated the expression of Krüppel-like factor-4 (KLF4) in VSMCs. KLF4 plays a key role in regulating VSMC phenotypic modulation. KLF4 antagonizes proliferation, facilitates migration, and down-regulates VSMC differentiation marker gene expression. We show that the VSMC differentiation marker genes smooth muscle alpha-actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD) are all down-regulated by CSA in VSMC monoculture, whereas cyclin-dependent kinase inhibitor-1A (CDKN1A) and matrix metalloproteinase-3 (MMP3) are up-regulated. CSA did not affect the abundance of the VSMC microRNA (MIR) markers MIR143 and MIR145. Administration of CSA to rat carotid artery in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, and smooth muscle myosin heavy chain (MYH11) mRNA levels. The tumor suppressor genes KLF4, p53, and CDKN1A, however, were up-regulated, as well as MMP3, MMP9, and collagen-VIII. CSA-treated arteries showed remarkable remodeling, including breakdown of the internal elastic lamina and reorientation of VSMCs, as well as increased KLF4 immunostaining in VSMCs and endothelial cells. Altogether, these data show that cyclosporin up-regulates KLF4 expression and promotes phenotypic modulation of VSMCs.
Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Fatores de Transcrição Kruppel-Like/biossíntese , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Antígenos de Diferenciação/metabolismo , Aorta/citologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Diferenciação Celular , Células Cultivadas , Ciclosporina/efeitos adversos , Regulação para Baixo , Imunossupressores/efeitos adversos , Fator 4 Semelhante a Kruppel , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Túnica Média/efeitos dos fármacos , Túnica Média/patologia , Regulação para CimaRESUMO
Myotilin (MYOT) is a promising candidate gene for Vocal Cord and Pharyngeal Weakness with Distal Myopathy (VCPDM, also known as MPD2). Located within the minimum VCPDM candidate interval, myotilin mutations also cause a similarly progressive and adult-onset muscle disease. We examined myotilin in VCPDM patients by sequence analysis, RT-PCR, Southern blotting, and western blotting. We detected no defects in the myotilin gene, transcript, or protein in VCPDM. We also report several useful SNPs and STRs for the analysis of myotilin in muscle diseases of suspected, yet unknown genetic origin. We conclude that MYOT mutations likely are not a cause of VCPDM.