RESUMO
Exchange protein directly activated by cAMP (Epac) mediates cAMP-mediated cell signal independent of protein kinase A (PKA). Mice lacking Epac1 displayed metabolic defect suggesting possible functional involvement of skeletal muscle and exercise capacity. Epac1 was highly expressed, but not Epac 2, in the extensor digitorum longus (EDL) and soleus muscles. The exercise significantly increased protein expression of Epac 1 in EDL and soleus muscle of wild-type (WT) mice. A global proteomics and pathway analyses revealed that Epac 1 deficiency mainly affected "the energy production and utilization" process in the skeletal muscle. We have tested their forced treadmill exercise tolerance. Epac1-/- mice exhibited significantly reduced exercise capacity in the forced treadmill exercise and lower number of type 1 fibers than WT mice. The basal protein level of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was reduced in the Epac1-/- mice. Furthermore, increasing expression of PGC-1α by exercise was also significantly attenuated in the skeletal muscle of Epac1-/- mice. The expressions of downstream target genes of PGC-1α, which involved in uptake and oxidation of fatty acids, ERRα and PPARδ, and fatty acid content were lower in muscles of Epac1-/-, suggesting a role of Epac1 in forced treadmill exercise capacity by regulating PGC-1α pathway and lipid metabolism in skeletal muscle. Taken together, Epac1 plays an important role in exercise capacity by regulating PGC-1α and fatty acid metabolism in the skeletal muscle.
Assuntos
Ácidos Graxos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Atividade Motora , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Estresse Fisiológico , Animais , Fatores de Troca do Nucleotídeo Guanina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiologia , Esforço FísicoRESUMO
BACKGROUND/AIMS: Breast cancer is a clinically and molecularly heterogeneous disease. Patients with triple-negative breast cancer (TNBC) have poorer outcomes than those with other breast cancer subtypes due to lack of effective molecular targets for therapy. The present study aimed to the identification of estrogen receptor (ER)ß as a novel mitochondrial target in TNBC cells, together with underlying mechanisms. METHODS: Expression of ERß in clinical breast samples were examined by qRT-PCR, immunohistochemistry and immunoblotting. Subcellular distribution and binding of ERß-Grp75 was determined by confocal microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. The effect of mitocondrial ERß(mitoERß) overexpression on cell proliferation and cell cycle distribution were assessed CCK-8 assays and FACS. Mitochondrial ROS, membrane potential, and Ca²âº level were measured using the specific fluorescent probes Mito-Sox, TMRE, and Rhod-2AM. The tumorigenic effect of mitoERß overexpression was assessed using an anchorage-independent growth assay, sphere formation and a mouse orthotopic xenograft model. RESULTS: ERß expression was lower in tumor tissue than in adjacent normal tissue of patients with breast cancer, and low levels of mitochondrial ERß (mitoERß) also were associated with increased tumor recurrence after surgery. Overexpression of mitoERß inhibited the proliferation of TNBC cells and tumor masses in an animal model. Moreover, overexpression of mitoERß increased ATP production in TNBC cells and normal breast MCF10A cells, with the latter completely reversed by mitoERß knockdown in MCF10A cells. Grp75 was found to positively regulate ERß translocation into mitochondria via a direct interaction. Coimmunoprecipitation and subcellular fractionation experiments revealed that ERß-Grp75 complex is stable in mitochondria. CONCLUSION: These results suggest that the up-regulation of mitoERß in TNBC cells ensures proper mitochondrial transcription, activating the OXPHOS system to produce ATP. Studying the effects of mitoERß on mitochondrial activity and specific mitochondrial gene expression in breast cancer might help predict tumor recurrence, inform clinical decision-making, and identify novel drug targets in the treatment of TNBC.
Assuntos
Trifosfato de Adenosina/biossíntese , Receptor beta de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Cálcio/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/metabolismo , Feminino , Corantes Fluorescentes/química , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Estadiamento de Neoplasias , Fosforilação Oxidativa , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metabolic disturbance and mitochondrial dysfunction are a hallmark of diabetic cardiomyopathy (DC). Resistance exercise (RE) not only enhances the condition of healthy individuals but could also improve the status of those with disease. However, the beneficial effects of RE in the prevention of DC and mitochondrial dysfunction are uncertain. Therefore, this study investigated whether RE attenuates DC by improving mitochondrial function using an in vivo rat model of diabetes. Fourteen Otsuka Long-Evans Tokushima Fatty rats were assigned to sedentary control (SC, n = 7) and RE (n = 7) groups at 28 weeks of age. Long-Evans Tokushima Otsuka rats were used as the non-diabetic control. The RE rats were trained by 20 repetitions of climbing a ladder 5 days per week. RE rats exhibited higher glucose uptake and lower lipid profiles, indicating changes in energy metabolism. RE rats significantly increased the ejection fraction and fractional shortening compared with the SC rats. Isolated mitochondria in RE rats showed increase in mitochondrial numbers, which were accompanied by higher expression of mitochondrial biogenesis proteins such as proliferator-activated receptor-γ coactivator-1α and TFAM. Moreover, RE rats reduced proton leakage and reactive oxygen species production, with higher membrane potential. These results were accompanied by higher superoxide dismutase 2 and lower uncoupling protein 2 (UCP2) and UCP3 levels in RE rats. These data suggest that RE is effective at ameliorating DC by improving mitochondrial function, which may contribute to the maintenance of diabetic cardiac contractility.
Assuntos
Cardiomiopatias Diabéticas/prevenção & controle , Metabolismo Energético , Mitocôndrias Musculares/metabolismo , Contração Miocárdica , Condicionamento Físico Animal/métodos , Animais , Cardiomiopatias Diabéticas/fisiopatologia , Metabolismo dos Lipídeos , Masculino , Ratos , Ratos Long-EvansRESUMO
Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and posttranslational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSP1 domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth.
Assuntos
Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/química , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Neurônios/metabolismo , Animais , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Neurônios/química , Neurônios/citologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Unlike stable and immobile cell line conditions, animal hearts contract and relax to pump blood throughout the body. Mitochondria play an essential role by producing biological energy molecules to maintain heart function. In this study, we assessed the effect of heart mimetic cyclic stretch on mitochondria in a cardiac cell line. To mimic the geometric and biomechanical conditions surrounding cells in vivo, cyclic stretching was performed on HL-1 murine cardiomyocytes seeded onto an elastic micropatterned substrate (10% elongation, 0.5â¯Hz, 4â¯h/day). Cell viability, semi-quantitative Q-PCR, and western blot analyses were performed in non-stimulated control and cyclic stretch stimulated HL-1â¯cell lines. Cyclic stretch significantly increased the expression of mitochondria biogenesis-related genes (TUFM, TFAM, ERRα, and PGC1-α) and mitochondria oxidative phosphorylation-related genes (PHB1 and CYTB). Western blot analysis confirmed that cyclic stretch increased protein levels of mitochondria biogenesis-related proteins (TFAM, and ERRα) and oxidative phosphorylation-related proteins (NDUFS1, UQCRC, and PHB1). Consequently, cyclic stretch increased mitochondrial mass and ATP production in treated cells. Our results suggest that cyclic stretch transcriptionally enhanced mitochondria biogenesis and oxidative phosphorylation without detrimental effects in a cultured cardiac cell line.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Biogênese de Organelas , Estresse Mecânico , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Expressão Gênica , Camundongos , Mitocôndrias Cardíacas/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/citologia , Fosforilação OxidativaRESUMO
Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca2+. To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 µM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 µM of Ech A with an IC50 for PKCι activity of 107 µM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low KD of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Naftoquinonas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Animais , Cálcio/metabolismo , Células Cultivadas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND & AIMS: Reagents designed to target cancer stem cells (CSCs) could reduce tumor growth, recurrence, and metastasis. We investigated the mitochondrial features of CSCs. METHODS: Colon adenocarcinoma fragments were obtained from 8 patients during surgery at Busan Paik Hospital in Korea. We used immunohistochemistry and quantitative polymerase chain reaction to compare expression of mitochondrial peroxiredoxin 3 (PRX3) in CD133(+)CD44(+) Lgr5(+)cells (CSCs) vs CD133(-)CD44(-)Lgr5(-) colon tumor cells (non-CSCs). Cell survival and expression of mitochondrial-related genes were analyzed in the presence of 5-fluorouracil and/or antimycin A. We used small-interfering and short-hairpin RNAs and an overexpression vector to study PRX3, which functions in the mitochondria. CD133(+) cells with PRX3 knockdown or overexpressing PRX3 were grown as xenograft tumors in immunocompromised mice. Metastasis was studied after injection of tumor cells in spleens of mice. We used chromatin immunoprecipitation and reporter assays to characterize transcriptional regulation of PRX3 by forkhead box protein 1. RESULTS: CSCs had a higher mitochondrial membrane potential and increased levels of adenosine triphosphate, Ca(2+), reactive oxygen species, and oxygen consumption than non-CSCs. Levels of PRX3 were increased in colon CSCs compared with non-CSCs. PRX3 knockdown reduced the viability of CSCs, but non non-CSCs, by inducing mitochondrial dysfunction. PRX3 knockdown reduced growth of CSCs as xenograft tumors or metastases in mice. The expression of FOXM1 activated transcription of PRX3 and expression of CD133 in colon CSCs. CONCLUSIONS: Human colon CSCs have increased mitochondrial function compared with colon tumor cells without stem cell properties. Colon CSCs overexpress the mitochondrial gene PRX3, which is required for maintenance of mitochondrial function and tumorigenesis, and is regulated by forkhead box protein 1, which also regulates expression of CD133 in these cells. These proteins might be therapeutic targets for colorectal cancer.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Neoplasias do Colo/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Peroxirredoxina III/metabolismo , Antígeno AC133 , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Cálcio/metabolismo , Sobrevivência Celular , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Metabolismo Energético , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HCT116 , Células HT29 , Humanos , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Consumo de Oxigênio , Peptídeos/genética , Peptídeos/metabolismo , Peroxirredoxina III/genética , Interferência de RNA , Terapêutica com RNAi , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Echinochrome A (Ech A), a marine bio-product isolated from sea urchin eggs, is known to have cardioprotective effects through its strong antioxidant and ATP-sparing capabilities. However, the effects of Ech A on cardiac excitation-contraction (E-C) are not known. In this study, we investigated the effects of Ech A on cardiac contractility and Ca(2+) handling in the rat heart. In ex vivo Langendorff hearts, Ech A (3 µM) decreased left ventricular developing pressure to 77.7 ± 6.5 % of basal level. In isolated ventricular myocytes, Ech A reduced the fractional cell shortening from 3.4 % at baseline to 2.1 %. Ech A increased both diastolic and peak systolic intracellular Ca(2+) ([Ca(2+)]i). However, the ratio of peak [Ca]i to resting [Ca]i was significantly decreased. Ech A did not affect the L-type Ca(2+) current. Inhibiting the Na(+)/Ca(2+) exchanger with either NiCl2 or SEA400 did not affect the Ech A-dependent changes in Ca(2+) handling. Our data demonstrate that treatment with Ech A results in a significant reduction in the phosphorylation of phospholamban at both serine 16 and threonine 17 leading to a significant inhibition of SR Ca(2+)-ATPase 2A (SERCA2A) and subsequent reduced Ca(2+) uptake into the intracellular Ca(2+) store. Taken together, our data show that Ech A negatively regulates cardiac contractility by inhibiting SERCA2A activity, which leads to a reduction in internal Ca(2+) stores.
Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/farmacologia , Miócitos Cardíacos/metabolismo , Naftoquinonas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Masculino , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Fosforilação , Ratos , Ratos Wistar , Serina/metabolismo , Treonina/metabolismo , Função VentricularRESUMO
Mutation or depletion of mitochondrial DNA (mtDNA) can cause severe mitochondrial malfunction, originating from the mitochondrion itself, or from the crosstalk between nuclei and mitochondria. However, the changes that would occur if the amount of mtDNA is diminished are less known. Thus, we generated rat myoblast H9c2 cells containing lower amounts of mtDNA via ethidium bromide and uridine supplementation. After confirming the depletion of mtDNA by quantitative PCR and gel electrophoresis analysis, we investigated the changes in mitochondrial physical parameters by using flow cytometry. We also evaluated the resistance of these cells to serum starvation and sodium nitroprusside. H9c2 cells with diminished mtDNA contents showed decreased mitochondrial membrane potential, mass, free calcium, and zinc ion contents as compared to naïve H9c2 cells. Furthermore, cytosolic and mitochondrial reactive oxygen species levels were significantly higher in mtDNA-lowered H9c2 cells than in the naïve cells. Although the oxygen consumption rate and cell proliferation were decreased, mtDNA-lowered H9c2 cells were more resistant to serum deprivation and nitroprusside insults than the naïve H9c2 cells. Taken together, we conclude that the low abundance of mtDNA cause changes in cellular status, such as changes in reactive oxygen species, calcium, and zinc ion levels inducing resistance to stress.
Assuntos
DNA Mitocondrial/genética , Dosagem de Genes , Miócitos Cardíacos/metabolismo , Nitroprussiato/metabolismo , Soro/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Potencial da Membrana Mitocondrial , Mitocôndrias/genética , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Estresse Oxidativo , Consumo de Oxigênio , Ratos , Espécies Reativas de OxigênioRESUMO
Neopetrosides A (1) and B (2), new naturally occurring ribosides of nicotinic acid with extremely rare α-N-glycoside linkages and residues of p-hydroxybenzoic and pyrrole-2-carboxylic acids attached to C-5', were isolated from a marine Neopetrosia sp. sponge. Structures 1 and 2 were determined by NMR and MS methods and confirmed by the synthesis of 1 and its ß-riboside analogue (3). Neopetroside A (1) upregulates mitochondrial functions in cardiomyocytes.
Assuntos
Nucleosídeos/química , Nucleosídeos/isolamento & purificação , Poríferos/química , Piridinas/química , Piridinas/isolamento & purificação , Trifosfato de Adenosina/análise , Animais , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Nucleosídeos/síntese química , Piridinas/síntese químicaRESUMO
B7-H4 is a B7 family coregulatory protein that inhibits T cell-mediated immunity. B7-H4 is overexpressed in various cancers; however, the functional role of B7-H4 in cancer metabolism is poorly understood. Because mitochondria play pivotal roles in development, proliferation, and death of cancer cells, we investigated molecular and functional alterations of mitochondria in B7-H4-depleted HeLa cells. In a human study, overexpression of B7-H4 was confirmed in the cervices of adenocarcinoma patients (n = 3) compared to noncancer patients (n = 3). In the cell line model, B7-H4 depletion was performed by transfection with small interfering RNA (siRNA). B7-H4 depletion suppressed oxygen consumption rate, ATP production, and mitochondrial membrane potential and mass and increased reactive oxygen species production. In particular, electron transport complex III activity was significantly impaired in siB7-H4-treated cells. Coincidently, depletion of B7-H4 suppressed major mitochondrial regulators (peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC1-α] and mitochondrial transcription factor A), a component of oxidative phosphorylation (ubiquinol-cytochrome c reductase core protein 1), and an antiapoptosis protein (Bcl-XL). Mitochondrial dysfunction in siRNA-treated cells significantly augmented oxidative stress, which strongly activated the JNK/P38/caspase axis in the presence of doxorubicin, resulting in increased apoptotic cell death. Investigating the mechanism of B7-H4-mediated mitochondrial modulation, we found that B7-H4 depletion significantly downregulated the cAMP/cAMP response element-binding protein/PGC1-α signaling pathway. Based on these findings, we conclude that B7-H4 has a role in the regulation of mitochondrial function, which is closely related to cancer cell physiology and drug sensitivity.
Assuntos
Adenocarcinoma/metabolismo , Regulação para Baixo , Mitocôndrias/metabolismo , Transdução de Sinais , Neoplasias do Colo do Útero/metabolismo , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismo , Idoso , Antibióticos Antineoplásicos/farmacologia , Apoptose , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Doxorrubicina/farmacologia , Feminino , Células HeLa , Humanos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/metabolismo , Inibidor 1 da Ativação de Células T com Domínio V-Set/genéticaRESUMO
Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neovascularização Patológica , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Compostos Bicíclicos com Pontes/química , Proteínas de Transporte/genética , Proliferação de Células , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Ácido Láctico/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação , Consumo de Oxigênio , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Echinochrome A (Ech A) is a natural pigment from sea urchins that has been reported to have antioxidant properties and a cardio protective effect against ischemia reperfusion injury. In this study, we ascertained whether Ech A enhances the mitochondrial biogenesis and oxidative phosphorylation in rat cardio myoblast H9c2 cells. To study the effects of Ech A on mitochondrial biogenesis, we measured mitochondrial mass, level of oxidative phosphorylation, and mitochondrial biogenesis regulatory gene expression. Ech A treatment did not induce cytotoxicity. However, Ech A treatment enhanced oxygen consumption rate and mitochondrial ATP level. Likewise, Ech A treatment increased mitochondrial contents in H9c2 cells. Furthermore, Ech A treatment up-regulated biogenesis of regulatory transcription genes, including proliferator-activated receptor gamma co-activator (PGC)-1α, estrogen-related receptor (ERR)-α, peroxisome proliferator-activator receptor (PPAR)-γ, and nuclear respiratory factor (NRF)-1 and such mitochondrial transcription regulatory genes as mitochondrial transcriptional factor A (TFAM), mitochondrial transcription factor B2 (TFB2M), mitochondrial DNA direct polymerase (POLMRT), single strand binding protein (SSBP) and Tu translation elongation factor (TUFM). In conclusion, these data suggest that Ech A is a potentiated marine drug which enhances mitochondrial biogenesis.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Renovação Mitocondrial/efeitos dos fármacos , Renovação Mitocondrial/genética , Mioblastos Cardíacos/efeitos dos fármacos , Naftoquinonas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Mioblastos Cardíacos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Echinochrome A (Ech A) is a naphthoquinoid pigment from sea urchins that possesses antioxidant, antimicrobial, anti-inflammatory and chelating abilities. Although Ech A is the active substance in the ophthalmic and cardiac drug Histochrome®, its underlying cardioprotective mechanisms are not well understood. In this study, we investigated the protective role of Ech A against toxic agents that induce death of rat cardiac myoblast H9c2 cells and isolated rat cardiomyocytes. We found that the cardiotoxic agents tert-Butyl hydroperoxide (tBHP, organic reactive oxygen species (ROS) inducer), sodium nitroprusside (SNP; anti-hypertension drug), and doxorubicin (anti-cancer drug) caused mitochondrial dysfunction such as increased ROS level and decreased mitochondrial membrane potential. Co-treatment with Ech A, however, prevented this decrease in membrane potential and increase in ROS level. Co-treatment of Ech A also reduced the effects of these cardiotoxic agents on mitochondrial oxidative phosphorylation and adenosine triphosphate level. These findings indicate the therapeutic potential of Ech A for reducing cardiotoxic agent-induced damage.
Assuntos
Cardiotônicos/farmacologia , Cardiotoxinas/antagonistas & inibidores , Mitocôndrias Cardíacas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Naftoquinonas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cardiotoxinas/toxicidade , Morte Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Ouriços-do-MarRESUMO
Ursolic acid (UA), a type of pentacyclic triterpenoid carboxylic acid purified from natural plants, can promote skeletal muscle development. We measured the effect of resistance training (RT) with/without UA on skeletal muscle development and related factors in men. Sixteen healthy male participants (age, 29.37±5.14 years; body mass index=27.13±2.16 kg/m(2)) were randomly assigned to RT (n=7) or RT with UA (RT+UA, n=9) groups. Both groups completed 8 weeks of intervention consisting of 5 sets of 26 exercises, with 10~15 repetitions at 60~80% of 1 repetition maximum and a 60~90-s rest interval between sets, performed 6 times/week. UA or placebo was orally ingested as 1 capsule 3 times/day for 8 weeks. The following factors were measured pre-and post-intervention: body composition, insulin, insulin-like growth factor-1 (IGF-1), irisin, and skeletal muscle strength. Body fat percentage was significantly decreased (p<0.001) in the RT+UA group, despite body weight, body mass index, lean body mass, glucose, and insulin levels remaining unchanged. IGF-1 and irisin were significantly increased compared with baseline levels in the RT+UA group (p<0.05). Maximal right and left extension (p<0.01), right flexion (p<0.05), and left flexion (p<0.001) were significantly increased compared with baseline levels in the RT+UA group. These findings suggest that UA-induced elevation of serum irisin may be useful as an agent for the enhancement of skeletal muscle strength during RT.
RESUMO
[This corrects the article on p. 441 in vol. 18, PMID: 25352765.].
RESUMO
Resveratrol is known to exert a cardioprotective effect against hypoxia/reoxygenation (H/R) injury. HS-1793 is a novel, more stable resveratrol analog, but its cardioprotective effects were unknown. The present study aimed to test the cardioprotective effect of HS-1793 against H/R injury and investigate the role of mitochondria in Sprague Dawley rat heart damage using an ex vivo Langendorff system. HS-1793 ameliorated H/R-induced mitochondrial dysfunction by reducing mitochondrial reactive oxygen species production, improving mitochondrial oxygen consumption and suppressing mitochondrial calcium (Ca(2+)) overload during reperfusion. Moreover, HS-1793-treated rat heart showed reduced infarct size. Our data suggest that HS-1793 can protect cardiac against mitochondrial damage following H/R, thereby suppressing injury.
Assuntos
Naftóis/química , Resorcinóis/química , Estilbenos/química , Animais , Cálcio/metabolismo , Coração/fisiopatologia , Hipóxia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Naftóis/farmacologia , Naftóis/uso terapêutico , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Resorcinóis/farmacologia , Resorcinóis/uso terapêutico , ResveratrolRESUMO
We investigated the impairment of ATP-sensitive K(+) (K(ATP)) channels in aortic smooth muscle cells (ASMCs) from isoproterenol-induced hypertrophied rabbits. The amplitude of K(ATP) channels induced by the K(ATP) channel opener pinacidil (10 µM) was greater in ASMCs from control than from hypertrophied animals. In phenylephrine-preconstricted aortic rings, pinacidil induced relaxation in a dose-dependent manner. The dose-dependent curve was shifted to the right in the hypertrophied (EC(50): 17.80 ± 3.28 µM) compared with the control model (EC(50): 6.69 ± 2.40 µM). Although the level of Kir6.2 subtype expression did not differ between ASMCs from the control and hypertrophied models, those of the Kir6.1 and SUR2B subtypes were decreased in the hypertrophied model. Application of the calcitonin-gene related peptide (100 nM) and adenylyl cyclase activator forskolin (10 µM), which activates protein kinase A (PKA) and consequently K(ATP) channels, induced a K(ATP) current in both control and hypertrophied animals; however, the K(ATP) current amplitude did not differ between the two groups. Furthermore, PKA expression was not altered between the control and hypertrophied animals. These results suggests that the decreased K(ATP) current amplitude and K(ATP) channel-induced vasorelaxation in the hypertrophied animals were attributable to the reduction in K(ATP) channel expression but not to changes in the intracellular signaling mechanism that activates the K(ATP) current.
Assuntos
Aorta/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Canais KATP/fisiologia , Músculo Liso Vascular/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Canais KATP/agonistas , Canais KATP/biossíntese , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Técnicas de Cultura de Órgãos , Pinacidil/farmacologia , Coelhos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
Although various stimuli-inducing cell demise are known to alter mitochondrial morphology, it is currently debated whether alteration of mitochondrial morphology is per se responsible for apoptosis execution or prevention. This study was undertaken to examine the effect of histone deacetylase (HDAC) inhibitors on mitochondrial fusion-fission equilibrium. The mechanism underlying HDAC inhibitor-induced alteration of mitochondrial morphology was examined in various cells including primary cultured cells and untransformed and cancer cell lines treated with seven different HDAC inhibitors. Suberoylanilide hydroxamic acid (SAHA)-induced mitochondrial elongation in both Hep3B and Bcl-2-overexpressing Hep3B cells, apart from its apoptosis induction function. SAHA significantly decreased the expression of mitochondrial fission protein Fis1 and reduced the translocation of Drp1 to the mitochondria. Fis1 overexpression attenuated SAHA-induced mitochondrial elongation. In addition, depletion of mitochondrial fusion proteins, Mfn1 or Opa1, by RNA interference also attenuated SAHA-induced mitochondrial elongation. All of the HDAC inhibitors we examined induced mitochondrial elongation in all the cell types tested at both subtoxic and toxic concentrations. These results indicate that HDAC inhibitors induce mitochondrial elongation, irrespective of the induction of apoptosis, which may be linked to alterations of mitochondrial dynamics regulated by mitochondrial morphology-regulating proteins. Since mitochondria have recently emerged as attractive targets for cancer therapy, our findings that HDAC inhibitors altered mitochondrial morphology may support the rationale for these agents as novel therapeutic approaches against cancer. Further, the present study may provide insight into a valuable experimental strategy for simple manipulation of mitochondrial morphology.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Acetilação/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Dinaminas , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Histonas/fisiologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , VorinostatRESUMO
Glucocorticoids (GCs) are essential steroid hormones for homeostasis, development, metabolism, and cognition and possess anti-inflammatory and immunosuppressive actions. Since glucocorticoid receptor II (GR) is nearly ubiquitous, chronic activation or depletion of GCs leads to dysfunction of diverse organs, including the heart and blood vessels, resulting predominantly from changes in gene expression. Most studies, therefore, have focused on the genomic effects of GC to understand its related pathophysiological manifestations. The nongenomic effects of GCs clearly differ from well-known genomic effects, with the former responding within several minutes without the need for protein synthesis. There is increasing evidence that the nongenomic actions of GCs influence various physiological functions. To develop a GC-mediated therapeutic target for the treatment of cardiovascular disease, understanding the genomic and nongenomic effects of GC on the cardiovascular system is needed. This article reviews our current understanding of the underlying mechanisms of GCs on cardiovascular diseases and stress, as well as how nongenomic GC signaling contributes to these conditions. We suggest that manipulation of GC action based on both GC and GR metabolism, mitochondrial impact, and the action of serum- and glucocorticoid-dependent kinase 1 may provide new information with which to treat cardiovascular diseases.