RESUMO
Several studies provide evidence that erythropoietin (EPO) could play an important role in the recovery of the heart subjected to ischemia-reperfusion. In this regard, it has been suggested that EPO could be involved in protein kinase B (Akt) activation as a cell survival protein. The aim of the present study was to investigate the effects of EPO on the Akt/glycogen synthase kinase 3 beta (GSK-3ß) pathway in the presence or absence of wortmannin (W, Akt inhibitor) and its relationship with mitochondrial morphology and function preservation in ischemic-reperfused rat hearts. EPO improved the functional recovery of the heart subjected to ischemia-reperfusion, reduced the release of CK and the infarct size, and promoted preservation of the mitochondrial structure. Moreover, it reduced tissue lactate content and preserved glycogen in order to prevent ischemia. The results showed greater Akt activation, accompanied by preservation of swelling and mitochondrial calcium retention capacity, as well as an increase in ATP synthesis capacity. These results were accompanied by an inhibition of GSK-3ß, suggesting regulation of Akt on the opening of the mitochondrial permeability transition pore. All these beneficial effects exerted by acute treatment with EPO were prevented by W. The present study provided novel evidence that EPO not only enhances intrinsic activation of Akt during myocardial ischemia-reperfusion but also promotes GSK-3ß inhibition, contributing to mitochondrial structure and function preservation.
Assuntos
Cardiotônicos , Eritropoetina , Coração , Proteínas Proto-Oncogênicas c-akt , Traumatismo por Reperfusão , Animais , Ratos , Eritropoetina/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Isquemia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Cardiotônicos/farmacologia , Coração/efeitos dos fármacosRESUMO
Recent studies have provided evidence that triiodothyronine (T3) might play an effective role in the recovery of ischemic myocardium, through the preservation of mitochondrial function and the improvement of energy substrate metabolism. To this respect, it has been suggested that T3 could activate AMP-activated protein kinase (AMPK), the cellular 'fuel-gauge' enzyme, although its role has yet to be elucidated. The aim of the present study was to investigate the effects produced by acute treatment with T3 (60 nM) and the pharmacological inhibition of AMPK by compound C on isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion. Results showed that T3 increased AMPK activation during simulated ischemia-reperfusion, while compound C prevented it. At the end of simulated reperfusion, acute T3 treatment increased contractile function recovery and cellular viability conservation. Mitochondrial ultrastructure was better preserved in the presence of T3 as well as mitochondrial ATP production rate and tissue ATP content. Calcium retention capacity, a parameter widely used as an indicator of the resistance of mitochondrial permeability transition pore (MPTP) to opening, and GSK-3ß phosphorylation, a master switch enzyme that limits MPTP opening, were increased by T3 administration. All these beneficial effects exerted by T3 acute treatment were prevented when compound C was co-administrated. The present study provided original evidence that T3 enhances intrinsic activation of AMPK during myocardial ischemia-reperfusion, being this enzyme involved, at least in part, in the protective effects exerted by T3, contributing to mitochondrial structure and function preservation, post-ischemic contractile recovery and conservation of cellular viability.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cardiotônicos/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , Miocárdio/patologia , Tri-Iodotironina/uso terapêutico , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cardiotônicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Diástole/efeitos dos fármacos , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Átrios do Coração/ultraestrutura , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Contração Miocárdica/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Sístole/efeitos dos fármacos , Tri-Iodotironina/farmacologiaRESUMO
The cardioprotective activity of rosuvastatin (R) is yet to be known. The objective of this study was to research whether R perfusion before global ischemia can mitigate myocardial ischemia-reperfusion damage, considering the metabolic condition in which these effects occur, and to contemplate potential mitochondrial benefits. Protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK-3ß) and mitochondrial permeability transition pore (MPTP) are key elements in myocardial injury produced by ischemia-reperfusion. Isolated rat hearts were subjected to 25-min ischemia and 1-h reperfusion in the presence or absence of R, with or without Wortmannin (W), a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor. Akt and GSK-3ß were measured by Western blot analysis; lactate, glycogen, and G6PDH were determined; and Ca2+-induced MPTP opening was evaluated using a spectrophotometric method. Contractility was assessed by left ventricular developed pressure (LVDP), and rate-pressure product (RPP), peak rate of contraction and peak rate of relaxation (± dP/dt), and left ventricular end-diastolic pressure (LVEDP) were determined. Tissue samples were extracted to evaluate mitochondrial damage by electron microscopy and to assess infarct size. Statistical analysis employed ANOVA (n = 6/per group). Myocardial infarct size was significantly reduced by R, which also improved cardiac function. MPTP opening was delayed to 300 µM CaCl2, while use of W resulted in MPTP opening at 200 µM CaCl2. Electron microscopy showed better mitochondrial preservation with R, which reduced lactic acid production, increased glycogen consumption and G6PDH activity, as well as phosphorylation of Akt and GSK-3ß. R before ischemia is cardioprotective against ischemic and reperfusion damage, activating Akt and regulating GSK-3ß negatively and attenuating the MPTP opening.
Assuntos
Cardiotônicos/uso terapêutico , Glicogênio Sintase Quinase 3 beta/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rosuvastatina Cálcica/uso terapêutico , Animais , Feminino , Coração/efeitos dos fármacos , Técnicas In Vitro , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Infarto do Miocárdio/patologia , Ratos , Ratos WistarRESUMO
El consumo crónico de alcohol en Uruguay es un problema creciente, sin embargo, las determinaciones de biomarcadores consensuados no se realizan sistemáticamente ni se investigan otros marcadores potenciales. Para validar la hipótesis de que las metaloproteinasas de matriz con actividad gelatinasa son biomarcadores de consumo crónico de alcohol, se evaluaron muestras de sangre de 100 alcohólicos que comenzaron a atenderse en la Unidad de Trastornos Relacionados con el Alcohol y de 50 donantes sanos no alcohólicos. Las muestras de alcohólicos presentaron actividad de gelatinasas que triplicaron la de los controles y aumentos pequeños pero significativos en los niveles de γ-glutamil transferasa, aspartato-aminotransferasa y volumen corpuscular medio. Los valores de transferrina deficiente en carbohidratos fueron menores en alcohólicos que en controles. Estos resultados permiten proponer a las gelatinasas como los indicadores más sensibles del consumo sostenido de alcohol en la población analizada, ya que las enzimas hepáticas y el volumen corpuscular medio muestran una tendencia acorde con la literatura pero no alcanzaron valores asociados a la patología. Dado que la transferrina deficiente en carbohidratos es considerada el biomarcador indirecto más sensible y específico de consumo crónico de alcohol, los valores menores obtenidos en alcohólicos respecto de controles sugieren problemas metodológicos que podrían subsanarse aplicando otras técnicas de medida o por la presencia de interferencias que deben ser identificadas. Finalmente, estos hallazgos justifican una extensión de este trabajo piloto, así como estudios adicionales centrados en la participación de las metaloproteinasas de matriz con actividad gelatinasa en las cascadas de daño asociadas al consumo crónico de alcohol.
Chronic alcohol consumption in Uruguay is a growing problem, however, determinations of consensual biomarker are not performed systematically neither potential markers are explored. To validate the hypothesis that matrix metalloproteinases with gelatinase activity are biomarkers of chronic alcohol consumption, blood samples of 100 alcoholics that began medical treatment at the Unidad de Trastornos Relacionados con el Alcohol and 50 healthy non-alcoholic donors were evaluated. Alcoholic samples showed gelatinase activity that tripled that of controls and small but significant increases in levels of γ-glutamyl transferase, aspartate-aminotransferase and mean cellular volume. Carbohydrate deficient transferrin values were lower in alcoholics than in controls. These results allow proposing gelatinases as the most sensitive indicators of sustained alcohol consumption in the population analyzed since hepatic enzymes and mean cellular volume showed a tendency consistent with the literature but did not reach values associated with the pathology. Since carbohydrate-deficient transferrin is considered the most sensitive and specific indirect biomarker of chronic alcohol consumption, lower values in alcoholics related to controls suggest methodological problems that could be solved by applying other measurement techniques or by the presence of yet unknown interferences. Finally, these findings justify an extension of this pilot work, as well as additional studies focused on the participation of matrix metalloproteinases with gelatinase activity in the cascades of damage associated with chronic alcohol consumption.
O consumo crônico de álcool no Uruguai é um problema crescente, no entanto, as determinações consensuais de biomarcadores não são realizadas sistematicamente ou os potenciais marcadores são explorados. Para validar a hipótese de que as metaloproteinases de matriz com atividade gelatinase são biomarcadores do consumo crônico de álcool, foram avaliadas amostras de sangue cd 100 alcoólatras que começaram a ser tratadas na Unidad de Trastornos Relacionados con el Alcohol e 50 doadores não-alcoólatras saudáveis. As amostras alcoólicas apresentaram atividade de gelatinase que triplicou a dos controles e pequenos más significativos aumentos nos níveis de γ-glutamil transferase, aspartato-aminotransferase e volume médio celular. Os valores de transferrina deficientes em carboidratos foram menores nos alcoolistas que nos controles. Esses resultados permitem que as gelatinases sejam propostas como os indicadores mais sensíveis do consumo sustentado de álcool na população analisada, uma vez que as enzimas hepáticas e o volume celular médio apresentam uma tendência consistente com a literatura, mas não alcançaram valores associados à patologia. Como a transferrina deficiente em carboidratos é considerada o biomarcador indireto mais sensível e específico do consumo crônico de álcool, os valores mais baixos em alcoólatras do que em controles sugerem problemas metodológicos que poderiam ser sanados pela aplicação de outras técnicas de mensuração pela presença de interferências que deben ser identificadas. Finalmente, esses achados justificam uma extensão deste trabalho piloto, bem como estudos adicionais voltados para a participação de metaloproteinases de matriz com atividade de gelatinase nas cascatas de danos associados ao consumo crônico de álcool.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Alcoolismo/diagnóstico , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Método Duplo-Cego , Estudos Transversais , Estudos de Coortes , Sensibilidade e Especificidade , Alcoolismo/enzimologia , Alcoolismo/sangue , Índices de Eritrócitos , gama-Glutamiltransferase/sangueRESUMO
AMP-activated protein kinase (AMPK) is a serine-threonine kinase that functions primarily as a metabolic sensor to coordinate anabolic and catabolic processes in the cell, via phosphorylation of multiple proteins involved in metabolic pathways, aimed to re-establish energy homeostasis at a cell-autonomous level. Myocardial ischemia and reperfusion represents a metabolic stress situation for myocytes. Whether AMPK plays a critical role in the metabolic and functional responses involved in these conditions remains uncertain. In this study, in order to gain a deeper insight into the role of endogenous AMPK activation during myocardial ischemia and reperfusion, we explored the effects of the pharmacological inhibition of AMPK on contractile function rat, contractile reserve, tissue lactate production, tissue ATP content, and cellular viability. For this aim, isolated atria subjected to simulated 75 min ischemia-75 min reperfusion (Is-Rs) in the presence or absence of the pharmacological inhibitor of AMPK (compound C) were used. Since in most clinical situations of ischemia-reperfusion the heart is exposed to high levels of fatty acids, the influence of palmitate present in the incubation medium was also investigated. The present results suggest that AMPK activity significantly increases during Is, remaining activated during Rs. The results support that intrinsic activation of AMPK has functional protective effects in the reperfused atria when glucose is the only available energetic substrate whereas it is deleterious when palmitate is also available. Cellular viability was not affected by either of these conditions.
Assuntos
Metabolismo Energético , Átrios do Coração/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Trifosfato de Adenosina/metabolismo , Animais , Função Atrial , Ácidos Graxos/metabolismo , Feminino , Glucose/metabolismo , Ácido Láctico/metabolismo , Contração Miocárdica , Ratos , Ratos Sprague-DawleyRESUMO
Ischemic preconditioning (IPC) is one of the most powerful interventions to reduce ischemia-reperfusion injury. The aim of the present study was to investigate the involvement of the phosphatidylinositol-3-kinases (PI3Ks) family in cardioprotection exerted by IPC and the relationship between preservation of mitochondrial morphology and ATP synthesis capacity. In this regard, macroautophagy (autophagy) is considered a dynamic process involved in the replacement of aged or defective organelles under physiological conditions. IPC consisted of four 5-min cycles of ischemia-reperfusion followed by sustained ischemia. Wortmannin (W), a PI3K family inhibitor, was added to the perfusion medium to study the involvement of autophagy in the beneficial effects of IPC. In the present study, LC3-II/I expression was significantly increased in the IPC group when compared with the control group. The hearts subjected to IPC showed greater degradation of p62 than control groups, establishing the existence of an autophagic flow. Electron microscopy showed that IPC preserves the structural integrity of mitochondria after ischemia and at the end of reperfusion. Moreover, hearts subjected to IPC exhibited increased mitochondrial ATP synthesis. The beneficial effects of IPC were abolished by W in all trials of this study, abolishing the differences between the IPC and control groups. These results suggest that IPC could partly reduce injury by ischemia-reperfusion (I/R) by decreasing mitochondrial damage and promoting autophagy. Since W is a nonspecific inhibitor of the PI3Ks family, further research is required to confirm participation of PI3K in the response to IPC.
Assuntos
Androstadienos/farmacologia , Cardiotônicos/farmacologia , Precondicionamento Isquêmico , Traumatismo por Reperfusão , Animais , Ratos , WortmaninaRESUMO
Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA). Isolated rat atria subjected to simulated 75-min ischaemia/75-min reperfusion (Is-Rs) in the presence or absence of 3-MA were used. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3-MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3-MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome-like structures during reperfusion, which were absent in atria subjected to Is-Rs in the presence of 3-MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is-Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to ß-adrenergic and to calcium stimulation at the end of Is-Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is-Rs in the presence of 3-MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia-reperfusion.