RESUMO
Myocardial infarction (MI) is one of the leading causes of high mortality worldwide. Long non-coding RNA myocardial infarction associated transcript (MIAT) and mitochondrial coupling factor 6 (CF6) aggravate MI. This study aimed to elucidate whether miR-203 interacted with MIAT and CF6 in MI. Results revealed that MIAT and CF6 expressions were upregulated and that miR-203 was downregulated in mouse myocardial tissues after MI, as well as in hypoxic mouse cardiomyocytes. The overexpression of MIAT in mouse cardiomyocytes raised CF6 expression, whereas the knockdown of MIAT had the opposite effect. Mechanistically, the luciferase reporter and RNA pull-down assays corroborated the binding between miR-203 and CF6 3'UTR and between miR-203 and MIAT. The simultaneous overexpression of miR-203 and MIAT restored the reduction of CF6 caused by miR-203 overexpression alone, and the overexpression of miR-203 diminished the percentage of infarct area and the apoptosis of cardiomyocytes in vivo. Our findings corroborate that overexpressing miR-203 alleviates MI via interacting with MIAT and CF6.
Assuntos
MicroRNAs/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Infarto do Miocárdio/genética , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Fatores Acopladores da Fosforilação Oxidativa/genética , RNA Longo não Codificante/genéticaRESUMO
Skeletal muscle mitochondrial respiration is thought to be altered in obesity, insulin resistance, and type 2 diabetes; however, the invasive nature of tissue biopsies is an important limiting factor for studying mitochondrial function. Recent findings suggest that bioenergetics profiling of circulating cells may inform on mitochondrial function in other tissues in lieu of biopsies. Thus, we sought to determine whether mitochondrial respiration in circulating cells [peripheral blood mononuclear cells (PBMCs) and platelets] reflects that of skeletal muscle fibers derived from the same subjects. PBMCs, platelets, and skeletal muscle (vastus lateralis) samples were obtained from 32 young (25-35 yr) women of varying body mass indexes. With the use of extracellular flux analysis and high-resolution respirometry, mitochondrial respiration was measured in intact blood cells as well as in permeabilized cells and permeabilized muscle fibers. Respiratory parameters were not correlated between permeabilized muscle fibers and intact PBMCs or platelets. In a subset of samples (n = 12-13) with permeabilized blood cells available, raw measures of substrate (pyruvate, malate, glutamate, and succinate)-driven respiration did not correlate between permeabilized muscle (per mg tissue) and permeabilized PBMCs (per 106 cells); however, complex I leak and oxidative phosphorylation coupling efficiency correlated between permeabilized platelets and muscle (Spearman's ρ = 0.64, P = 0.030; Spearman's ρ = 0.72, P = 0.010, respectively). Our data indicate that bioenergetics phenotypes in circulating cells cannot recapitulate muscle mitochondrial function. Select circulating cell bioenergetics phenotypes may possibly inform on overall metabolic health, but this postulate awaits validation in cohorts spanning a larger range of insulin resistance and type 2 diabetes status.
Assuntos
Células Sanguíneas/metabolismo , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Consumo de Oxigênio/fisiologia , Adulto , Glicemia/análise , Plaquetas/metabolismo , Índice de Massa Corporal , Metabolismo Energético/fisiologia , Feminino , Humanos , Insulina/sangue , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Triglicerídeos/sangueRESUMO
Diet-induced metabolic acidosis is associated with the impairment of bone metabolism and an increased risk of a number of chronic noncommunicable diseases, such as type 2 diabetes mellitus and hypertension. Low serum bicarbonate is associated with high mortality in healthy older individuals. Recently, we demonstrated that both coupling factor 6 (CF6)-overexpressing transgenic (TG) and high salt-fed mice which had sustained intracellular acidosis, due to enhanced proton import through ecto-F1Fo complex and/or reduced proton export through Na+-K+ ATPase inhibition, displayed shortened lifespan and early senescence-associated phenotypes such as signs of hair greying and alopecia, weight loss, and/or reduced organ mass. In this study, we searched causative genes of proton-induced aging in CF6-overexpressing TG and high salt-fed mice. We discovered NM_026333 as a novel anti-aging gene which was downregulated in the heart and kidney in both types of mice. NM_026333 protein consists of 269 amino acids with transmembrane region (90-193aa). Induction of NM_026333 or recombinant protein rescued TG cells and CF6-treated human cells from aging hallmarks of impaired autophagy, genomic instability, and epigenetic alteration. NM_026333 protein directly bound plasma membrane Na+-Ca2+ exchanger 1 (NCX1) to suppress its reverse mode, and cancelled proton-induced epigenetic regression of Atg7 that was caused by H3K4 and H4K20 tri-methylation via suppression of demethylase and H4K5 acetylation via suppression of nuclear HDAC3-HDAC4-emerin system. NM_026333 also attenuated proton-induced impaired formation of autolysosome, an increase in nuclear acetylated LC3 II, and acetylation of Atg7. These effects reappeared by NCX1 inhibitor. Furthermore, NCX1 inhibitor extended lifespan compared with vehicle-treatment in TG mice. This study will shed light on novel aging mechanism and provide implications in a target for anti-aging therapy.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Autofagia/genética , Autofagia/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Epigenômica , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Células HEK293 , Humanos , Camundongos , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Prótons , Transdução de Sinais/efeitos dos fármacosRESUMO
Diet-induced metabolic acidosis is associated with the impairment of bone metabolism and an increased risk of a number of chronic noncommunicable diseases, such as type 2 diabetes mellitus and hypertension. The serum bicarbonate level is an independent predictor of chronic kidney disease progression. We investigated whether proton accelerates aging by analyzing both coupling factor 6-overexpressing transgenic (TG) and high salt-fed mice which display sustained intracellular acidosis, due to enhanced proton import through ecto-F1 Fo complex and/or reduced proton export through Na+ -K+ ATPase inhibition. Both types of mice displayed shortened lifespan and early senescence-associated phenotypes such as signs of hair greying and alopecia, weight loss, and/or reduced organ mass. In chronic intracellular acidosis mice, autophagy was impaired by regression of Atg7, an increase in nuclear acetylated LC3 II, and acetylation of Atg7. The increase in histone 3 trimethylation at lysine 4 (H3K4me3) and H4K20me3 and the decrease in H3K9me3 and H3K27me3 were observed in the heart and kidney obtained from both TG and high salt-fed mice. The decrease in lamin A/C, emerin, and heterochromatin protein 1α without changes in barrier-to-autointegration factor and high-mobility group box 1 was confirmed in TG and high salt-fed mice. Suppression of nuclear histone deacetylase 3-emerin system is attributable to epigenetic regression of Atg7 and H4K5 acetylation. These findings will shed light on novel aging and impaired autophagy mechanism, and provide implications in a target for antiaging therapy.
Assuntos
Envelhecimento/fisiologia , Epigênese Genética , Instabilidade Genômica , Acidose/etiologia , Animais , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Pressão Sanguínea , Montagem e Desmontagem da Cromatina , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Prótons , Cloreto de Sódio na Dieta/farmacologiaRESUMO
Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex, resulting in proton import and accelerated aging. Inhibitory factor peptide 1 (IF1) suppresses a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis. We tested the hypothesis that IF1 may attenuate CF6-induced aging signaling in CF6-overexpressing transgenic (TG) cells. In IF1-GFP overexpressing wild type (WT) cells, the diffuse peripheral staining of tubular mitochondria was observed with a dense widely distributed network around the nucleus. In TG cells, however, the only peri-nuclear network of fragmented mitochondria was observed at 24 h, but it was developed to a widely distributed mitochondrial network of tubular mitochondria at 72 h. TG cells displayed aging hallmarks of telomere attrition, epigenetic alterations, defective proteostasis, and genomic instability with a decrease in emerin and lamin and loss of heterochromatin. IF1 induction rescued TG cells from telomere attrition, expression of genomic instability with the increase in emerin and lamin, and that of epigenetic alterations with recovery of heterochromatin. In defective proteostasis, IF1 induction restored a potent peri-nuclear staining of autolysosomes compared with the baseline weak staining. The decrease in Atg7 was restored, whereas the increase in P62 was abolished. We conclude that genetic disruption of proton signals by IF1 induction suppressed CF6-induced expression of aging hallmarks such as telomere attrition, epigenetic alterations, defective proteostasis, and genomic instability. Given the widespread biological actions of CF6, the physiological and pathological actions of IF1 may be complex.
Assuntos
Envelhecimento , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Proteínas/metabolismo , Animais , Instabilidade Genômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Proteínas/genética , Proteína Inibidora de ATPaseRESUMO
The incidence and development of colorectal cancer (CRC) is a process with multiple gene interactions. We have previously demonstrated that ATP synthase-coupling factor 6, mitochondrial (ATP5J) is associated with CRC migration and 5-fluorouracil resistance; nevertheless, the exact molecular mechanism remains unclear. The following study uses microarray and bioinformatics methods to identify candidate genes and long non-coding RNAs (lncRNAs) in CRC cells (two pairs) with upregulated and downregulated ATP5J. Briefly, a total of 2,190 differentially expressed mRNAs (DEmRNAs) were sorted. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed for 4 DEmRNAs to validate the results of microarray analysis. Functional annotation and pathway enrichment were analyzed for DEmRNAs using the Database for Annotation, Visualization and Integrated Discovery. Significantly enriched pathways included the regulation of gene expression and cell growth. The proteinprotein interaction network was constructed, and AKT serine/threonine kinase 2 (AKT2) was considered as one of the hub genes. For further analysis, 51 DEmRNAs and 30 DElncRNAs were selected that were positively or negatively associated with the expression of ATP5J in the two cell pairs. X-inactive specific transcript (XIST), premature ovarian failure 1B (POF1B) and calmin (CLMN) were identified in the DEmRNA-DElncRNA co-expression network. The expression of AKT2 and XIST in CRC cells was confirmed by RT-qPCR. To sum up, the candidate genes and lncRNAs, as well as potential signaling pathways, which were identified using integrated bioinformatics analysis, could improve the understanding of molecular events involved in the function of ATP5J in CRC.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Proteínas Proto-Oncogênicas c-akt/biossíntese , RNA Longo não Codificante/biossíntese , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Humanos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , RNA Longo não Codificante/genéticaRESUMO
The present study aimed to elucidate the role of marine collagen peptides (MCPs) in protection of carotid artery vascular endothelial cells (CAVECs) in type 2 diabetes mellitus (T2DM), and the mechanism underlying this process. In an in vivo experiment, diabetic Wistar rats were divided randomly into four groups (n=10/group): Diabetes control, and three diabetes groups administered low, medium and high doses of MCPs (2.25, 4.5 and 9.0 g/kg body weight/day, respectively). Another 10 healthy rats served as the control. In an in vitro experiment, human umbilicalvein endothelial cells (HUVECs) were incubated in normal and high concentrations of glucose with or without MCPs (3.0, 15.0 and 30.0 mg/ml, respectively) for 24, 48 or 72 h. Blood vessel/endothelial construction, inflammatory exudation and associated molecular biomarkers in CAVECs were detected and analyzed. The results of the present study demonstrated that in rats, MCP treatment for 4 weeks significantly lowered blood glucose and attenuated endothelial thinning and inflammatory exudation in carotidartery vascular endothelial cells. In vitro, the highglucose intervention significantly increased cell apoptosis in HUVECs, and medium and high doses of MCPs (4.5 and 9.0 g/kg body weight/day, respectively) partially ameliorated this high glucosemediated apoptosis and decreased levels of apoptosis biomarkers. In conclusion, a moderate oral MCP dose (≥4.5 g/kg body weight/day) may be a novel therapeutic tool to protect against early cardiovascular complications associated with T2DM by inhibiting apoptosis and reducing the expression of coupling factor 6 and microparticles.
Assuntos
Apoptose/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Colágeno/química , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Peptídeos/farmacologia , Animais , Organismos Aquáticos/química , Biomarcadores , Glicemia , Células Cultivadas , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Peptídeos/química , RatosRESUMO
High sodium, high glucose, and obesity are important risk factors for age-related diseases such as cardiovascular disease (CVDs), stroke, and cancer. Coupling factor 6 (CF6) is released from vascular endothelial cells and functions as a circulating peptide that inhibits prostacyclin and nitric oxide generation by intracellular acidosis. High glucose elevates CF6 by activation of protein kinase C and p38 mitogen-activated protein kinase, whereas CF6 causes type 2 diabetes mellitus, resulting in a high glucose vicious cycle. Low glucose increases inhibitory factor peptide 1, an endogenous inhibitor of CF6. High salt intake increases CF6 through nuclear factor κB signaling, whereas CF6 induces salt-sensitive hypertension and salt-induced congestive heart failure. Oral administration of vitamin C cancels salt-induced increase in CF6, and estrogen replacement leads to the delayed onset of CF6-induced salt-sensitive hypertension and the rescue from cardiac systolic dysfunction. Because CF6 contributes to the onset of CVDs, nutritional regulation of CF6 will shed light on the understanding of preventive strategy and mechanisms for CVDs and a target for therapy.
Assuntos
Células Endoteliais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Administração Oral , Ácido Ascórbico/farmacologia , Diabetes Mellitus Tipo 2/diagnóstico , Epoprostenol/antagonistas & inibidores , Epoprostenol/metabolismo , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/etiologia , Humanos , Hipertensão/diagnóstico , NF-kappa B/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Cloreto de Sódio na Dieta/administração & dosagem , Cloreto de Sódio na Dieta/efeitos adversos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
To investigate the effect of excessive fluoride on the mitochondrial function of cardiomyocytes, 20 healthy male mice were randomly divided into 2 groups of 10, as follows: control group (animals were provided with distilled water) and fluoride group (animals were provided with 150 mg/L F- drinking water). Ultrastructure and pathological morphological changes of myocardial tissue were observed under the transmission electron and light microscopes, respectively. The content of hydrolysis ATP enzyme was observed by ATP enzyme staining. The expression levels of ATP5J and ATP5H were measured by Western blot and quantitative real-time PCR. The morphology and ultrastructure of cardiomyocytes mitochondrial were seriously damaged by fluoride, including the following: concentration of cardiomyocytes and inflammatory infiltration, vague myofilaments, and mitochondrial ridge. The damage of mitochondrial structure was accompanied by the significant decrease in the content of ATP enzyme for ATP hydrolysis in the fluoride group. ATP5J and ATP5H expressions were significantly increased in the fluoride group. Thus, fluoride induced the mitochondrial dysfunction in cardiomyocytes by damaging the structure of mitochondrial and interfering with the synthesis of ATP. The proactive ATP5J and ATP5H expression levels were a good response to the mitochondrial dysfunction in cardiomyocytes.
Assuntos
Fluoretos/toxicidade , Mitocôndrias Cardíacas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Trifosfato de Adenosina , Animais , Fluoretos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Mitocôndrias Cardíacas/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Fatores Acopladores da Fosforilação Oxidativa/genéticaRESUMO
Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on de novo translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. PTCD1, ATP5J2-PTCD1, CADPS2, and STPG1 were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike de novo translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.
Assuntos
Pontos de Quebra do Cromossomo , Transtornos Cromossômicos/genética , Mapeamento Cromossômico/métodos , Fenótipo , Translocação Genética/genética , Adolescente , Adulto , Proteínas de Ligação ao Cálcio/genética , Pré-Escolar , Transtornos Cromossômicos/diagnóstico , Feminino , Genoma Humano , Humanos , Masculino , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Linhagem , Análise de Sequência de DNA/métodos , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare and fatal disease. The prostacyclin (PGI2) pathway is a well-known therapeutic target for PAH. As a novel vasoconstrictive peptide, coupling factor 6 (CF6) has been recognized as the only endogenous inhibitor of PGI2. However, the relationship between CF6 and PAH is still unknown. In this study, we investigated the involvement of CF6 in PAH in rats. METHODS: A total of 80 Sprague-Dawley rats were randomly divided into 2 groups: a control group (with saline intraperitoneally) and a monocrotaline (MCT)-treated group (with MCT 60mg/kg intraperitoneal injection). The expression level of CF6 was measured by immunohistochemistry, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The plasma level of 6-keto-PGF1α, a stable metabolite of PGI2, was measured by enzyme-linked immunosorbent assay. RESULTS: After MCT injection, although the plasma level of CF6 was markedly increased in a time-dependent manner, the level of 6-keto-PGF1α was decreased in the MCT rats as compared to that in the controls (P < 0.05). Reverse transcription polymerase chain reaction indicated that the lung tissue level of CF6 messenger RNA (mRNA) in the MCT rats was significantly upregulated compared to the level in the controls. There was an intense CF6 immunostain concentrated on the lung tissue of the MCT rats compared with the controls. Univariate analysis indicated that the plasma level of CF6 was not correlated with blood pressure, fasting blood glucose, cholesterol (high-density lipoprotein, triglycerides and total cholesterol) or C-reactive protein level. CONCLUSIONS: These results demonstrated that CF6 was elevated in MCT-induced PAH rats and may play an important role in the development of PAH.
Assuntos
Epoprostenol/sangue , Hipertensão Pulmonar/sangue , ATPases Mitocondriais Próton-Translocadoras/sangue , Fatores Acopladores da Fosforilação Oxidativa/sangue , Animais , Hemodinâmica , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/fisiopatologia , Imuno-Histoquímica , Monocrotalina , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease associated with high morbidity and mortality rates. However, the exact regulatory mechanism of PAH is unknown. Although coupling factor 6 (CF6) is known to function as a repressor, its role in PAH has not been explored. Here, we investigated the involvement of endogenous CF6 in the development of PAH. METHODS: PAH was induced with monocrotaline (MCT), as demonstrated by significant increases in pulmonary artery pressure and vessel wall thickness. The adeno-associated virus (AAV) carrying CF6 short hairpin RNA (shRNA) or control vector (2×10(10) gp) was intratracheally transfected into the lungs of rats 2 weeks before or after MCT injection. RESULTS: A 2-6-fold increase in CF6 was observed in the lungs and circulation of the MCT-injected rats as confirmed by qRT-PCR and ELISA. Immunohistochemistry analysis revealed a small quantity of CF6 localized to endothelial cells (ECs) under physiological conditions spread to surrounding tissues in a paracrine manner in PAH lungs. Notably, CF6 shRNA effectively inhibited CF6 expression, abolished lung macrophage infiltration, reversed endothelial dysfunction and vascular remodeling, and ameliorated the severity of pulmonary hypertension and right ventricular dysfunction at 4 weeks both as a pretreatment and rescue intervention. In addition, the circulating and lung levels of 6-keto-PGF1a, a stable metabolite of prostacyclin, were reversed by CF6 inhibition, suggesting that the effect of CF6 inhibition may partly be mediated through prostacyclin. CONCLUSIONS: CF6 contributes to the pathogenesis of PAH, probably in association with downregulation of prostacyclin. The blockage of CF6 might be applied as a novel therapeutic approach for PAH and PA remodeling.
Assuntos
Terapia Genética/métodos , Hipertensão Pulmonar/terapia , Pulmão/metabolismo , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/antagonistas & inibidores , Fatores Acopladores da Fosforilação Oxidativa/genética , Interferência de RNA , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Hipertensão Pulmonar/induzido quimicamente , Injeções Espinhais , Pulmão/patologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Monocrotalina , Infiltração de Neutrófilos , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Artéria Pulmonar/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Remodelação Vascular , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/genética , Disfunção Ventricular Direita/prevenção & controleRESUMO
Cardiovascular health is influenced by dietary composition and the western diet is composed of varying types/amounts of fat. Conjugated linoleic acid (cLA) is an abundant dietary unsaturated fatty acid associated with health benefits but its biological signaling is not well understood. Nitrite is enriched in vegetables within the diet and can impact signaling of unsaturated fatty acids; however, its role on cLA signaling is not well understood. Elucidating how nitrite may impact the biological signaling of cLA is important due to the dietary consumption of both cLA and nitrite in the western diet. Since co-administration of cLA and nitrite results in cardioprotection during myocardial infarction (MI), it was hypothesized that cLA and nitrite may affect cardiac mitochondrial respiratory function and complex activity in MI. C57BL/6J mice were treated with cLA and nitrite for either 10 or 13days, where MI was induced on day 3. Following treatment, respiration and complex activity were measured. Among the major findings of this study, cLA treatment (10days) decreases state 3 respiration in vivo. Following MI, nitrite alone and in combination with cLA attenuates increased state 3 respiration and decreases hydrogen peroxide levels. Further, nitrite and cLA co-treatment attenuates increased complex III activity after MI. These results suggest that cLA, nitrite and the combination significantly alter cardiac mitochondrial respiratory and electron transport chain activity in vivo and following MI. Overall, the daily consumption of cLA and nitrite in the diet can have diverse cardiovascular implications, some of which occur at the mitochondrial level.
Assuntos
Cardiotônicos/uso terapêutico , Suplementos Nutricionais , Modelos Animais de Doenças , Ácidos Linoleicos Conjugados/uso terapêutico , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Nitrito de Sódio/uso terapêutico , Animais , Cardiotônicos/administração & dosagem , Ecocardiografia , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Coração/diagnóstico por imagem , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Ácidos Linoleicos Conjugados/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Estresse OxidativoRESUMO
Glioblastoma (GBM) is the most common primary malignant brain tumor. Microvascular proliferation is one of the characteristic pathologic features of GBM. Mitochondrial dysfunction plays an important role in the pathogenesis of GBM. In this study, microvascular proliferation from GBM and normal brain blood vessels were laser microdissected and total RNA was isolated from these microvasculatures. The difference of mRNA expression profiles among GBM microvasculature, normal brain blood vessels and GBM tumor cells was evaluated by mitochondria and metabolism PCR gene arrays. It was found that the mRNA levels of ATP5A1 and ATP5B in GBM tumor cells as well as microvascular proliferation were significantly higher compared with normal brain blood vessels. Immunohistochemical stains with anti-ATP5A1 antibody or anti-ATP5B antibody were performed on tissue microarray, which demonstrated strongly positive expression of ATP5A1 and ATP5B in GBM tumor cells and GBM microvascular proliferation while normal blood vessels were negative. By analyzing The Cancer Genome Atlas data sets for GBM and other cancers, genomic DNA alterations (mutation, amplification or deletion) were less likely the reason for the high expression of ATP5A1 and ATP5B in GBM. Our miRNA microarray data showed that miRNAs that target ATP5A1 or ATP5B were down-regulated, which might be the most likely reason for the high expression of ATP5A1 and ATP5B in GBM tumor cells and microvascular proliferation. These findings help us better understand the pathogenesis of GBM, and agents against ATP5A1 and/or ATP5B might effectively kill both tumor cells and microvascular proliferation in GBM. MiRNAs, such as Let-7f, miR-16, miR-23, miR-100 and miR-101, that target ATP5A1 or ATP5B, might be potential therapeutic agents for GBM.
Assuntos
Neoplasias Encefálicas/genética , Endotélio Vascular/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Microvasos/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Endotélio Vascular/citologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Técnicas Imunoenzimáticas , MicroRNAs/genética , Microvasos/citologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , Células Tumorais CultivadasRESUMO
Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex unlike a proton-mediated clockwise rotation in the mitochondria, resulting in ATP hydrolysis, proton import, and apoptosis. Inhibitory peptide 1 (IF1) inhibits a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis by a clockwise rotation. We tested the hypothesis that IF1 may antagonize the biological action of CF6 in human embryonic kidney 293 cells. We generated mature and immature IF1 expression vectors and those labeled with GFP at the C-terminus. In the immature IF1-GFP overexpressing cells, the mitochondrial network of IF1-GFP was newly found at the plasma membrane after peripheral translocation, whereas in mature IF1-GFP transfected cells, a less punctuate rather homogenous pattern was found in the cytoplasm. IF1 protein was detected in the exosome fraction of culture media, and it was enhanced by mature or immature IF1 transfection. Extracellular ATP hydrolysis was enhanced by CF6, whereas immature or mature IF1 transfection suppressed ATP hydrolysis in response to CF6. Intracellular pH was decreased by CF6 but was unchanged after immature IF1 transfection. CF6-induced increase in apoptotic cells was blocked by immature or mature IF1, being accompanied by protein kinase B (PKB) phosphorylation. IF1 antagonizes the pro-apoptotic action of CF6 by relief of intracellular acidification and resultant phosphorylation of PKB. Given the widespread biological actions of CF6, the physiological and pathological functions of IF1 may be expected to be complex. J. Cell. Biochem. 117: 1680-1687, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Apoptose , Exossomos/metabolismo , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/antagonistas & inibidores , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Proteínas/metabolismo , Exossomos/genética , Células HEK293 , Humanos , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Fosforilação/genética , Transporte Proteico/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Transfecção , Proteína Inibidora de ATPaseRESUMO
Mitochondrial ribosomes synthesize a subset of hydrophobic proteins required for assembly of the oxidative phosphorylation complexes. This process requires temporal and spatial coordination and regulation, so quality control of mitochondrial protein synthesis is paramount to maintain proteostasis. We show how impaired turnover of de novo mitochondrial proteins leads to aberrant protein accumulation in the mitochondrial inner membrane. This creates a stress in the inner membrane that progressively dissipates the mitochondrial membrane potential, which in turn stalls mitochondrial protein synthesis and fragments the mitochondrial network. The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism that we propose acts as a sensor to couple the synthesis of mitochondrial proteins with organelle fitness, thus ensuring coordinated assembly of the oxidative phosphorylation complexes from two sets of ribosomes.
Assuntos
Proteases Dependentes de ATP/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/patologia , Proteínas Mitocondriais/biossíntese , Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Linhagem Celular , Membrana Celular/fisiologia , Células HEK293 , Humanos , Ácidos Hidroxâmicos/farmacologia , Potencial da Membrana Mitocondrial/fisiologia , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Fosforilação Oxidativa , Fatores Acopladores da Fosforilação Oxidativa/biossíntese , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Interferente PequenoRESUMO
Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aß-treated cells. Indeed, Aß bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aß-induced impairment in ATP production and ATPase activity. These results indicate that Aß-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aß and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A.
Assuntos
Doença de Alzheimer/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Acetilglucosamina/metabolismo , Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Animais , Células CHO , Cricetulus , Modelos Animais de Doenças , Glicosilação , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Fatores Acopladores da Fosforilação Oxidativa/genética , Processamento de Proteína Pós-Traducional , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
BACKGROUND: Our recent study has demonstrated that inhibition of calpain by transgenic overexpression of calpastatin reduces myocardial proinflammatory response and dysfunction in endotoxemia. However, the underlying mechanisms remain to be determined. In this study, we used cardiomyocyte-specific capn4 knockout mice to investigate whether and how calpain disrupts ATP synthase and induces mitochondrial superoxide generation during endotoxemia. METHODS AND RESULTS: Cardiomyocyte-specific capn4 knockout mice and their wild-type littermates were injected with lipopolysaccharides. Four hours later, calpain-1 protein and activity were increased in mitochondria of endotoxemic mouse hearts. Mitochondrial calpain-1 colocalized with and cleaved ATP synthase-α (ATP5A1), leading to ATP synthase disruption and a concomitant increase in mitochondrial reactive oxygen species generation during lipopolysaccharide stimulation. Deletion of capn4 or upregulation of ATP5A1 increased ATP synthase activity, prevented mitochondrial reactive oxygen species generation, and reduced proinflammatory response and myocardial dysfunction in endotoxemic mice. In cultured cardiomyocytes, lipopolysaccharide induced mitochondrial superoxide generation that was prevented by overexpression of mitochondria-targeted calpastatin or ATP5A1. Upregulation of calpain-1 specifically in mitochondria sufficiently induced superoxide generation and proinflammatory response, both of which were attenuated by ATP5A1 overexpression or mitochondria-targeted superoxide dismutase mimetics. CONCLUSIONS: Cardiomyocyte-specific capn4 knockout protects the heart against lipopolysaccharide-induced injury in endotoxemic mice. Lipopolysaccharides induce calpain-1 accumulation in mitochondria. Mitochondrial calpain-1 disrupts ATP synthase, leading to mitochondrial reactive oxygen species generation, which promotes proinflammatory response and myocardial dysfunction during endotoxemia. These findings uncover a novel mechanism by which calpain mediates myocardial dysfunction in sepsis.
Assuntos
Calpaína/genética , Endotoxemia/complicações , Regulação da Expressão Gênica , Mitocôndrias Cardíacas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/genética , Animais , Calpaína/biossíntese , DNA/genética , Modelos Animais de Doenças , Endotoxemia/genética , Endotoxemia/metabolismo , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/patologia , ATPases Mitocondriais Próton-Translocadoras/biossíntese , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Fatores Acopladores da Fosforilação Oxidativa/biossínteseRESUMO
BACKGROUND: Mitochondrial coupling factor 6 (CF6) is a constriction factor in cardiac hypertrophy, whose mechanisms are not fully understood. MATERIALS AND METHODS: Here, we established cardiac hypertrophy models for feeding spontaneously hypertensive rats (SHRs) aged 10, 20, and 30 weeks. Hemodynamic monitoring was performed during the feeding program to ensure the success of the model. RESULTS: Cardiac hypertrophy, but not fibrosis, occurred in the 10-, 20-, and 30-week-old SHRs. No significant changes in CF6 gene expression were detected by RT-PCR in any of the SHR groups as compared with the control groups (p > 0.05). ELISA assessment showed that the CF6 protein level in the 20- and 30-week-old SHRs with cardiac hypertrophy was significantly increased (vs. control, p < 0.05). CONCLUSION: CF6 protein was upregulated in cardiac hypertrophy induced by hypertension; further mechanisms involved in this process should be investigated.
Assuntos
Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Hipertensão/complicações , Hipertensão/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fatores Acopladores da Fosforilação Oxidativa/metabolismo , Animais , Biomarcadores/metabolismo , Masculino , Ratos Endogâmicos SHR , Ratos WistarRESUMO
Downhill skiing in the elderly increases maximal oxygen uptake (VO2max) and carbohydrate handling, and produces muscle hypertrophy. We hypothesized that adjustments of the cellular components of aerobic glucose combustion in knee extensor muscle, and cardiovascular adjustments, would increase in proportion to VO2max. Nineteen healthy elderly subjects (age 67.5 ± 2.9 years) who completed 28.5 days of guided downhill skiing over 3 months were assessed for anthropometric variables, cardiovascular parameters (heart rate, hematocrit), VO2max, and compared with controls (n = 20). Biopsies of vastus lateralis muscle were analyzed for capillary density and expression of respiratory chain markers (NDUFA9, SDHA, UQCRC1, ATP5A1) and the glucose transporter GLUT4. Statistical significance was assessed with a repeated analysis of variance and Fisher's post-hoc test at a P value of 5%. VO2max increased selectively with ski training (+7 ± 2%). Capillary density (+11 ± 5%) and capillary-to-fiber ratio (12 ± 5%), but not the concentration of metabolic proteins, in vastus lateralis were increased after skiing. Cardiovascular parameters did not change. Fold changes in VO2max and capillary-to-fiber ratio were correlated and were under genetic control by polymorphisms of the regulator of vascular tone, angiotensin converting enzyme. The observations indicate that increased VO2max after recreational downhill ski training is associated with improved capillarity in a mainly recruited muscle group.