RESUMO
Mitochondrial ATP-sensitive K+ channels (mitoKATP) have been recently characterized structurally, and possess a protein through which K+ enters mitochondria (MitoKIR), and a regulatory subunit (mitoSUR). The mitoSUR regulatory subunit is an ATP-binding cassette (ABC) protein isoform 8 (ABCB8). Opening these channels is known to be cardioprotective, but the molecular and physiological mechanisms that activate them are not fully known. Here, to better understand the molecular and physiological mechanisms of activators (GTP) and inhibitors (ATP) on the activity of mitoKATP, we exposed isolated mitochondria to both nucleotides. We also used molecular docking directed to the nucleotide-binding domain of human ABCB8/mitoSUR to test a comparative model of ATP and GTP effects. As expected, we find that ATP dose-dependently inhibits mitoKATP activity (IC50 = 21.24 ± 1.4 µM). However, simultaneous exposure of mitochondria to GTP dose-dependently (EC50 = 13.19 ± 1.33 µM) reversed ATP inhibition. Pharmacological and computational studies suggest that GTP reverses ATP activity competitively. Docking directed to the site of crystallized ADP reveals that both nucleotides bind to mitoSUR with high affinity, with their phosphates directed to the Mg2+ ion and the walker A motif of the protein (SGGGKTT). These effects, when combined, result in GTP binding, ATP displacement, mitochondrial ATP-sensitive K+ transport, and lower formation of reactive oxygen species. Overall, our findings demonstrate the basis for ATP and GTP binding in mitoSUR using a combination of biochemical, pharmacological, and computational experiments. Future studies may reveal the extent to which the balance between ATP and GTP actions contributes toward cardioprotection against ischemic events.
Assuntos
Trifosfato de Adenosina , Canais KATP , Humanos , Canais KATP/metabolismo , Simulação de Acoplamento Molecular , Trifosfato de Adenosina/metabolismo , Mitocôndrias , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Potássio/metabolismoRESUMO
Typically, healthy cardiac tissue utilizes more fat than any other organ. Cardiac hypertrophy induces a metabolic shift leading to a preferential consumption of glucose over fatty acids to support the high energetic demand. Calorie restriction is a dietary procedure that induces health benefits and lifespan extension in many organisms. Given the beneficial effects of calorie restriction, we hypothesized that calorie restriction prevents cardiac hypertrophy, lipid content changes, mitochondrial and redox dysregulation. Strikingly, calorie restriction reversed isoproterenol-induced cardiac hypertrophy. Isolated mitochondria from hypertrophic hearts produced significantly higher levels of succinate-driven H2O2 production, which was blocked by calorie restriction. Cardiac hypertrophy lowered mitochondrial respiratory control ratios, and decreased superoxide dismutase and glutathione peroxidase levels. These effects were also prevented by calorie restriction. We performed lipidomic profiling to gain insights into how calorie restriction could interfere with the metabolic changes induced by cardiac hypertrophy. Calorie restriction protected against the consumption of several triglycerides (TGs) linked to unsaturated fatty acids. Also, this dietary procedure protected against the accumulation of TGs containing saturated fatty acids observed in hypertrophic samples. Cardiac hypertrophy induced an increase in ceramides, phosphoethanolamines, and acylcarnitines (12:0, 14:0, 16:0, and 18:0). These were all reversed by calorie restriction. Altogether, our data demonstrate that hypertrophy changes the cardiac lipidome, causes mitochondrial disturbances, and oxidative stress. These changes are prevented (at least partially) by calorie restriction intervention in vivo. This study uncovers the potential for calorie restriction to become a new therapeutic intervention against cardiac hypertrophy, and mechanisms in which it acts.
Assuntos
Restrição Calórica , Lipidômica , Cardiomegalia/induzido quimicamente , Cardiomegalia/tratamento farmacológico , Cardiomegalia/prevenção & controle , Humanos , Peróxido de Hidrogênio/metabolismo , Isoproterenol/metabolismo , Isoproterenol/toxicidade , Mitocôndrias/metabolismo , Oxirredução , Estresse OxidativoRESUMO
Mitochondria dysfunction is an important factor involved in PD pathogenesis. We reported neuroprotective actions of vitamin D (VD3) on a PD model, and now we investigated the VD3 effects on the brain mitochondrial function. We focused on oxygen consumption, respiratory control ratio (RCR), ADP/O ratio, mitochondria swelling, H2O2 production, and SOD activity. Additionally, immunohistochemistry assays for the dopamine system markers (TH and DAT) and mitochondrial markers (VDAC1 and Hsp60) were also carried out in the striata. Young adult male Wistar rats (250 g, 2.5 months age) were anesthetized and subjected to stereotaxic surgery and injection of saline (SO group) or 6-OHDA, into the right striatum. Brain mitochondria were isolated from the groups: sham-operated (SO), 6-OHDA, 6-OHDA pretreated with VD3 for 7, days before the 6-OHDA lesion (6-OHDA+VD3, pre-) or treated with VD3 for 14 days, after the 6-OHDA lesion (6-OHDA+VD3, post-). VD3 prevented decreases in oxygen consumption, RCR, and ADP/O ratio observed after 6-OHDA injury. Noteworthy, a very low (oxygen consumption and RCR) or no improvement (ADP/O) were observed in the 6-OHDA+VD3 post- group. VD3 also prevented the increased mitochondria swelling and H2O2 production and a decrease in SOD activity, respectively, in the 6-OHDA injured mitochondria. Also, VD3 supplementation protected the hemiparkinsonian brain from decreases in TH and DAT expressions and decreased the upregulation of mitochondrial markers, as VDAC 1 and Hsp60. In conclusion, VD3 showed neuroprotective actions on brain mitochondria injured by 6-OHDA and should stimulate translational studies focusing on its use as a therapeutic strategy for the treatment of neurodegenerative diseases as PD.
Assuntos
Doença de Parkinson , Animais , Encéfalo/metabolismo , Suplementos Nutricionais , Modelos Animais de Doenças , Peróxido de Hidrogênio/farmacologia , Masculino , Mitocôndrias/metabolismo , Estresse Oxidativo , Oxidopamina/toxicidade , Doença de Parkinson/metabolismo , Ratos , Ratos Wistar , Vitamina D/farmacologiaRESUMO
Mitochondrial ATP-sensitive potassium channels (mitoKATP) locate in the inner mitochondrial membrane and possess protective cellular properties. mitoKATP opening-induced cardioprotection (using the pharmacological agent diazoxide) is preventable by antagonists, such as glibenclamide. However, the mechanisms of action of these drugs and how mitoKATP respond to them are poorly understood. Here, we show data that reinforce the existence of a mitochondrial sulfonylurea receptor (mitoSUR) as part of the mitoKATP. We also show how diazoxide and glibenclamide compete for the same binding site in mitoSUR. A glibenclamide analog that lacks its cyclohexylurea portion (IMP-A) loses its ability to inhibit diazoxide-induced swelling. These results suggest that the cyclohexylureia portion of glibenclamide is indispensable for mitoKATP inhibition. Moreover, IMP-A did not suppress diazoxide-induced preconditioning (EC50 10.66 µM) in a rat model of a cardiac ischemia/reperfusion. Importantly, glibenclamide inhibited both diazoxide-induced cardioprotection (IC50 86 nM). We suggest that IMP-A must be used with caution since we found this drug possesses significant inhibitory effects on mitochondrial respiration. We characterized the binding of glibenclamide and diazoxide using a molecular simulation (docking) approach. Using the molecular structure of the ATP binding protein ABCB8 (pointed by others as the mitoSUR) we demonstrate that glibenclamide competitively inhibits diazoxide actions. This was reinforced (pharmacologically) in a competitive antagonism test. Taken together, these results bring valuable and novel insights into the pharmacological/biochemical aspects of mitokATP activation and cardioprotection. This study may lead to the discovery of novel therapeutic strategies that may impact ischemia-reperfusion injury.
Assuntos
Diazóxido , Canais KATP , Animais , Glibureto , Simulação de Acoplamento Molecular , RatosRESUMO
Oxidative stress, characterized by the accumulation of reactive oxygen species (ROS), is implicated in the pathogenesis of several diseases, including cardiac hypertrophy. The flavonoid quercetin is a potent ROS scavenger, with several beneficial effects for the cardiovascular system, including antihypertrophic effects. Oxidative imbalance has been implicated in cardiac hypertrophy and heart failure. In this work, we tested whether quercetin could attenuate cardiac hypertrophy by improving redox balance and mitochondrial homeostasis. To test this hypothesis, we treated a group of mice with isoproterenol (30 mg/kg/day) for 4 or 8 consecutive days. Another group received quercetin (10 mg/kg/day) from day 5th of isoproterenol treatment. We carried out the following assays in cardiac tissue: measurement of cardiac hypertrophy, protein sulfhydryl, catalase, Cu/Zn and Mn-superoxide dismutase (SOD) activity, detection of H2O2, and opening of the mitochondrial permeability transition pore. The animals treated with isoproterenol for the initial 4 days showed increased cardiac weight/tibia length ratio, decreased protein sulfhydryl content, compromised SOD and catalase activity, and high H2O2 levels. Quercetin was able to attenuate cardiac hypertrophy, restore protein sulfhydryl, and antioxidant activity, in addition to efficiently blocking the H2O2. We also observed that isoproterenol decreases mitochondrial SOD activity, while quercetin reverses it. Strikingly, quercetin also protects mitochondria against the opening of mitochondrial permeability transition pore. Taken together, these results suggest that quercetin is capable of reversing established isoproterenol-induced cardiac hypertrophy through the restoration of cellular redox balance and protecting mitochondria.
Assuntos
Antioxidantes/uso terapêutico , Cardiomegalia/tratamento farmacológico , Quercetina/uso terapêutico , Animais , Antioxidantes/farmacologia , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Isoproterenol , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Quercetina/farmacologia , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Cardiac hypertrophy involves marked wall thickening or chamber enlargement. If sustained, this condition will lead to dysfunctional mitochondria and oxidative stress. Mitochondria have ATP-sensitive K+ channels (mitoKATP) in the inner membrane that modulate the redox status of the cell. OBJECTIVE: We investigated the in vivo effects of mitoKATP opening on oxidative stress in isoproterenol- induced cardiac hypertrophy. METHODS: Cardiac hypertrophy was induced in Swiss mice treated intraperitoneally with isoproterenol (ISO - 30 mg/kg/day) for 8 days. From day 4, diazoxide (DZX - 5 mg/kg/day) was used in order to open mitoKATP (a clinically relevant therapy scheme) and 5-hydroxydecanoate (5HD - 5 mg/kg/day) or glibenclamide (GLI - 3 mg/kg/day) were used as mitoKATP blockers. RESULTS: Isoproterenol-treated mice had elevated heart weight/tibia length ratios (HW/TL). Additionally, hypertrophic hearts had elevated levels of carbonylated proteins and Thiobarbituric Acid Reactive Substances (TBARS), markers of protein and lipid oxidation. In contrast, mitoKATP opening with DZX avoided ISO effects on gross hypertrophic markers (HW/TL), carbonylated proteins and TBARS, in a manner reversed by 5HD and GLI. Moreover, DZX improved mitochondrial superoxide dismutase activity. This effect was also blocked by 5HD and GLI. Additionally, ex vivo treatment of isoproterenol- induced hypertrophic cardiac tissue with DZX decreased H2O2 production in a manner sensitive to 5HD, indicating that this drug also acutely avoids oxidative stress. CONCLUSION: Our results suggest that diazoxide blocks oxidative stress and reverses cardiac hypertrophy. This pharmacological intervention could be a potential therapeutic strategy to prevent oxidative stress associated with cardiac hypertrophy.
Assuntos
Cardiomegalia/tratamento farmacológico , Diazóxido/uso terapêutico , Peróxido de Hidrogênio/metabolismo , Canais de Potássio/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Diazóxido/farmacologia , Avaliação Pré-Clínica de Medicamentos , Transporte de Íons/efeitos dos fármacos , Isoproterenol/toxicidade , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Potássio/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
The purpose of this meta-analysis is to evaluate the association of Epstein-Barr virus (EBV) with oral squamous cell carcinoma (OSCC). We searched the electronic scientific databases of PubMed and Scopus and included a total of 53 studies that were published from 1990 to 2019. The analysis yielded a 45.37% (95% confidence interval [CI]: 38.90-51.84; p < 0.001) overall pooled prevalence of EBV. Studies that used the applied methods of in situ hybridization, polymerase chain reaction, immunology, or RNA microarray showed the following pooled prevalence: 46.08%, 40.32, 54.97%, and 74.89%, respectively. EBV-infected individuals have a 2.5 higher risk for developing OSCC (odds ratio: 2.57; 95% CI: 1.23% to 5.36%; p < 0.001). The present meta-analysis supports the hypothesis of EBV association with OSCC, pointing to this virus as a risk factor for neoplasia. Our findings also suggest that EBV latent transcripts (latent membrane protein 1, EBV nuclear antigen 1 and 2, and EBV-encoded small RNAs) have an important role in this process. Furthermore, novel advancements could arise from large and standardized studies that are constructed to probe for other latent gene expression, eliminate confounding factors (tobacco, alcohol, and high-risk human papillomavirus infection), and define the relationship between EBV and oral carcinomas.
Assuntos
Carcinoma de Células Escamosas/fisiopatologia , Infecções por Vírus Epstein-Barr/fisiopatologia , Herpesvirus Humano 4/fisiologia , Neoplasias Bucais/fisiopatologia , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/epidemiologia , Consumo de Bebidas Alcoólicas/fisiopatologia , Carcinoma de Células Escamosas/epidemiologia , Carcinoma de Células Escamosas/virologia , Infecções por Vírus Epstein-Barr/epidemiologia , Humanos , Neoplasias Bucais/epidemiologia , Neoplasias Bucais/virologia , Fumar/efeitos adversos , Fumar/epidemiologia , Fumar/fisiopatologiaRESUMO
OBJECTIVES: Human papillomavirus (HPV) has been implicated as a major factor in cervical carcinogenesis. However, many pieces of evidence gathered over the last two decades suggest Epstein-Barr virus (EBV) plays a secondary role in this process. The purpose of the present meta-analysis was to determine whether the presence of EBV infection increases the risk of cervical carcinoma. METHODS: Based on 25 articles, the analysis yielded a 33.44% overall pooled prevalence of EBV. RESULTS: The pooled prevalence was higher in patients with carcinoma (43.63%) than in healthy patients (19.0%) or patients with cervical intraepithelial neoplasia 1 (CIN1) (27.34%) or CIN2/3 (34.67%). Co-infection with EBV and HPV displayed a similar pattern. EBV infection was significantly and positively associated with lesion grade in cervical epithelia and was more prevalent in malignant lesions. Moreover, cervical carcinoma occurred four times as often among EBV positive women as in women without EBV infection (OR=4.01 [1.87-8.58]; p<0.001). CONCLUSIONS: The existence of EBV(+)HPV(-) carcinomas, the confirmed expression of latent oncoproteins (EBNA1, EBNA2, LMP1) and EBERs in tumor cells, and the association of EBV with the integration of high-risk-HPV DNA in malignant specimens point to EBV as a co-factor (so far underestimated) in the genesis and/or progression of cervical carcinoma. However, further studies are necessary before the link between EBV and cervical carcinoma can be established.