Complejo I, H2O2 y NO mitocondriales como señales prodrómicas de la disfunción cardíaca en diabetes tipo 1 / Mitochondrial Complex I, H2O2 and NO as Prodromal Signals of Cardiac Dysfunction in Type 1 Diabetes

RESUMEN Introducción: Resultados de nuestro laboratorio sugieren que la disfunción mitocondrial en el corazón precede a la falla miocárdica asociada a la hiperglucemia sostenida. Objetivo: Estudiar los eventos tempranos que ocurren en las mitocondrias de corazón en un modelo de diabetes mellitus tipo 1. Materiales y métodos: Ratas Wistar macho fueron inyectadas con estreptozotocina (STZ; 60 mg/kg, ip) y sacrificadas 10 o 14 días posinyección. Se obtuvo la fracción mitocondrial de corazón. Resultados: El consumo de O2 en estado 3 en presencia de malato-glutamato (21%) o succinato (16%) y las actividades de los complejos I-III (27%), II-III (24%) y IV (22%) fueron menores en los animales diabéticos a los 14 días posinyección. Cuando los animales se sacrificaron al día 10, solo el consumo de O2 en estado 3 en presencia de sustratos del complejo I (23%) y su control respiratorio (30%) fueron menores en las ratas inyectadas con STZ, de acuerdo con una reducción en la actividad del complejo I-III (17%). Estos cambios se acompañaron de un aumento en las velocidades de producción de H2O2 (117%), NO (30%) y ONOO- (∼225%), en la expresión de mtNOS (29%) y en la [O2 -]ss (∼150%) y [NO]ss (∼30%), junto con una disminución de la actividad de la Mn-SOD (15%) y la [GSSG+GSH]mitocondrial (28%), sin cambios en la expresión de PGC-1α. Conclusión: La disfunción del complejo I y el aumento en la generación de H2O2, NO y ONOO- pueden considerarse señales subcelulares prodrómicas del deterioro de la función mitocondrial que precede a la disfunción cardíaca en la diabetes.

ABSTRACT Background: Previous results from our laboratory suggest that heart mitochondrial dysfunction precedes myocardial failure associated with sustained hyperglycemia. Purpose: The aim of this study was to analyze the early events that take place in heart mitochondria in a type 1 diabetes mellitus (DM) model. Methods: Male Wistar rats were injected with streptozotocin (STZ; 60 mg/kg, ip.) to induce DM. They were euthanized 10 or 14 days later and the heart mitochondrial fraction was obtained. Results: State 3 O2 consumption in the presence of malate-glutamate (21%) or succinate (16%), and complex I-III (27%), II-III (24%) and IV (22%) activities were lower in diabetic animals 14 days after STZ injection. When animals were euthanized at day 10, only state 3 O2 consumption sustained by complex I substrates (23%) and its corresponding respiratory control (30%) were lower in rats injected with STZ, in agreement with reduced complex I-III activity (17%). These changes were accompanied by increased H2O2 (117%), NO (30%) and ONOO- (~225%) production rates, mtNOS expression (29%) and O2 - (~150%) and NO (~30%) steady-state concentrations, together with a decrease in Mn-SOD activity (15%) and mitochondrial [GSSG+GSH] (28%), without changes in PGC-1α expression. Conclusion: Complex I dysfunction and increased H2O2, NO and ONOO- production rates can be considered subcellular prodromal signals of the mitochondrial damage that precedes myocardial dysfunction in diabetes.

Comparison of the phenolic contents and epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.)

Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct­producing cultivars

Papel de la tiorredoxina-1 en el atontamiento cardíaco en ratones transgénicos / Role of Thioredoxin-1 on Myocardial Stunning in Transgenic Mice

Introducción: La disfunción ventricular posisquémica (miocardio atontado) involucra un aumento del estrés oxidativo. En este sentido, la célula cuenta con mecanismos de defensa, como la tiorredoxina-1, un antioxidante que protege al miocardio de la lesión por isquemia/reperfusión, reduciendo el tamaño del infarto. Objetivo: Evaluar el comportamiento de la función ventricular sistólica y diastólica, particularmente estudiando la rigidez miocárdica y la relajación isovolúmica en el miocardio atontado en diferentes ratones transgénicos. Material y métodos: Se utilizaron corazones de ratones que sobreexpresan tiorredoxina-1 y de ratones transgénicos que sobreexpresan tiorredoxina-1 mutada en su sitio activo (dominante negativo), comparados con los de ratones no transgénicos, los cuales fueron sometidos a 15 minutos de isquemia global y 30 minutos de reperfusión utilizando la técnica de Langendorff. Se evaluó la función ventricular sistólica y diastólica y se calculó el t63 y el t93 como índice de relajación isovolúmica. Resultados: Las mediciones a los 30 minutos de reperfusión mostraron una mejoría significativa del estado contráctil en los ratones tiorredoxina-1 (57,4 ± 4,9 mm Hg; p ≤ 0,05 vs. no transgénicos) y también en la rigidez (11,8 ± 2,9 mm Hg; p ≤ 0,05 vs. no transgénicos). Por otra parte, en los ratones dominantes negativos se observó un aumento de la rigidez (37,7 ± 5,5 mm Hg; p ≤ 0,05 vs. no transgénicos) y un enlentecimiento de la relajación a los 30 minutos de la reperfusión (78,2 ± 9,8 mseg; p ≤ 0,05 vs. no transgénicos). Conclusión: Este trabajo evidencia el rol protector de la tiorredoxina-1 en el miocardio atontado y su importancia fisiopatológica en ratones que sobreexpresan este antioxidante.

Background: Postischemic ventricular dysfunction (myocardial stunning) involves increased oxidative stress. In this sense, the cell has defense mechanisms, as thioredoxin-1, an antioxidant that protects the myocardium from ischemia/reperfusion injury, reducing infarct size. Objective: The aim of this study was to evaluate systolic and diastolic ventricular function, specifically analyzing myocardial stiffness and isovolumic relaxation, during myocardial stunning in different transgenic mice. Methods: Hearts from mice overexpressing thioredoxin-1 and transgenic mice overexpressing thioredoxin-1 with gene mutation in its active site (dominant negative) were compared with hearts from non-transgenic mice after 15-minute global ischemia and 30-minute reperfusion using the Langendorff technique. Systolic and diastolic ventricular function was evaluated and t63 and t93 were calculated as ventricular relaxation index. Results: At 30-minute reperfusion, thioredoxin-1 mice showed a significantly improved contractile state (57.4±4.9 mmHg; p≤0.05 vs. non-transgenic mice) and stiffness (11.8±2.9 mmHg; p≤0.05 vs. non-transgenic mice). Conversely, at the same reperfusion time, dominant negative mice exhibited increased stiffness (37.7±5.5 mmHg; p≤0.05 vs. non-transgenic mice) and slower relaxation (78.2±9.8 ms; p≤0.05 vs. non-transgenic mice). Conclusion: This study reveals the protective role of thioredoxin-1 on myocardial stunning and its pathophysiological importance in mice overexpressing this antioxidant.

Shock hemorrágico: óxido nítrico en ratas anestesiadas y no anestesiadas / Hemorrhagic Shock: Nitric Oxide in Anesthetized and Non Anesthetized Rats

Antecedentes En un trabajo previo mostramos que el estado hipovolémico inducido por una pérdida aguda de sangre se acompaña de una activación dinámica, heterogénea y dependiente del tiempo de la óxido nítrico sintetasa (NOS) cardíaca. Este sistema estaría involucrado en las alteraciones hemodinámicas que se observan luego de la depleción de volumen sanguíneo. Objetivo El objetivo del presente trabajo fue evaluar la participación del sistema del óxido nítrico (NO) mitocondrial en la respuesta adaptativa del sistema cardiovascular ante un shock hipovolémico en ratas anestesiadas y no anestesiadas. Material y métodos El estudio se llevó a cabo con cuatro grupos de animales (n = 7 por grupo): grupo A, ratas control anestesiadas; grupo C, ratas control no anestesiadas; grupo AH, ratas anestesiadas sometidas a una hemorragia (20% de la volemia) y grupo CH, ratas no anestesiadas sometidas a una hemorragia. Se evaluaron el consumo de oxígeno, la actividad funcional de la NOS mitocondrial (mtNOS) y la producción mitocondrial de NO. Resultados No se observaron diferencias significativas entre los valores de control respiratorio en los distintos grupos estudiados. La actividad funcional de la mtNOS fue menor en el grupo AH respecto del grupo A (12 ± 2 y 19 ± 1, respectivamente). Este efecto fue de menor magnitud cuando la hemorragia se provocó en animales no anestesiados (17 ± 1 y 20 ± 1, respectivamente). La producción mitocondrial de NO disminuyó en los grupos sometidos a una pérdida aguda de sangre, tanto no anestesiados como anestesiados, respecto de los animales controles. Conclusiones El sistema del NO mitocondrial estaría involucrado en la respuesta de adaptación del sistema cardiovascular frente a la depleción aguda de volumen. Esta participación dependería del grado de anestesia del animal.

Background We have previously demonstrated that hypovolemia induced by acute bleeding is accompanied by a dynamic, heterogenous and time-dependent activation of the cardiac nitric oxide synthase (NOS). This system might be involved in the hemodynamic anomalies observed after blood volume depletion. Objective To assess the role of the mitochondrial nitric oxide (NO) system in the adaptive response of the cardiovascular system in anesthetized and non anesthetized rats under hypovolemic shock. Material and Methods Animals were divided in four groups (n=7 animals per group): Group A, anesthetized control rats; group C, non anesthetized control rats; group AB, anesthetized rats subjected to bleeding (20% of blood volume), and group CB, non anesthetized rats subjected to bleeding. Oxygen consumption, functional activity of mitochondrial NOS (mtNOS) and mitochondrial production of NO were assessed. Results There were no significant differences in the values of respiratory parameters among the different study groups. Group AB had less functional activity of mtNOS compared to group A (12±2 and 19±1, respectively). This effect was even lower in non anesthetized animals subjected to bleeding (17±1 and 20±1, respectively). Mitochondrial production of NO decreased in anesthetized and non anesthetized animals with acute bleeding compared to controls. Conclusions Mitochondrial NO system might be involved in the adaptive response of the cardiovascular system under acute volume depletion, depending on the animal's degree of anesthesia.

Variability of antioxidant activity among honeybee-collected pollen of different botanical origin

Se determinaron las actividades antioxidantes de los extractos crudos de una mezcla de polen apícola y de cada uno de los seis polen constituyentes que formaban esa mezcla. Las determinaciones se hicieron por el método de sustancias reactivas al ácido tiobarbitúrico (TBARS) en preparaciones microsomales de hígado y por el método del bloqueo del radical libre 2,2-difenil-1-picrilhidracilo (DPPH*). Las actividades se compararon con la composición de flavonoles y ácidos fenólicos y con los contenidos de flavonoles en el polen. Todos los extractos mostraron actividades antioxidantes. Éstas fueron diferentes para cada especie y no estuvieron claramente asociadas al contenido de flavonoles en el polen. El polen de Amarathus hybridus mostró una alta capacidad inhibidora de la oxidación lipídica. El de la mezcla entera y el de Tagetes sp. fueron efectivos bloqueadores de radicales libres.

The antioxidant activities of total extracts of a mixture of honeybee-collected pollen and its six constituent pollens were determined by lipid peroxidation assay by the thiobarbituric acid reactive substances (TBARS) test on hepatic microsomal preparations and by free radical scavenging (2,2-diphenyl-1-picrylhydrazyl; DPPH*) method. Activities were compared to the flavonol and phenolic acid compositions and flavonol contents in pollen. All extracts showed antioxidant activities as radical scavenger substances and as inhibitors of lipid peroxidation. Antioxidant activities were different for each species and were not clearly associated to the flavonol content in pollen. Pollen of Amaranthus hybridus was a potent lipid oxidation inhibitor, and that of Tagetes sp. and the whole mixture were effective antiradical substances.

Determinaram-se as atividades antioxidantes dos extratos crus de uma mescla de pólen apícola e de cada um dos seis polens constituintes que formavam essa mescla. As determinações se fizeram pelo método de substâncias reativas ao ácido tiobarbitúrico (TBARS) em preparações microssomais de fígado e pelo método do bloqueio do radical livre 2,2-difenil-1-picrilhidracilo (DPPH*). As atividades se compararam com a composição de flavonóis e ácidos fenólicos e com os conteúdos de flavonóis no pólen. Todos os extratos mostraram atividades antioxidantes. Estas foram diferentes para cada espécie e não estiveram claramente associadas ao conteúdo de flavonóis no pólen. O pólen de Amarathus hybridus mostrou uma alta capacidade inibidora da oxidação lipídica. O da mescla inteira e o de Tagetes sp. foram efetivos bloqueadores de radicais livres.

Polyphenols and red wine as antioxidants against peroxynitrite and other oxidants

The antioxidant capacity of polyphenols (+)-catechin, (-)-epicatechin and myricetin, and of different types of red wines (Cabernet Sauvignon, Malbec and blended wine) was evaluated by three assays. (a) NADH oxidation by peroxynitrite (ONOO-): the ONOO- scavenging activity was higher for myricetin (IC50=35 µM) than for (+)-catechin (IC50=275 µM) and (-)-epicatechin (IC50=313 µM). (b) Peroxynitrite initiated chemiluminescence in rat liver homogenate: (-)-epicatechin (IC50=7.0 µM) and (+)-catechin (IC50=13 µM) were more potent than myricetin (IC50=20 µM) in inhibiting the chemiluminescence signal. (c) Lucigenin chemiluminescence in aortic rings: (-)-epicatechin (IC50=15 µM) and (+)-catechin (IC50=18 µM) showed higher antioxidant capacity than myricetin (IC50=32 µM). All the assayed red wines were able to scavenge the oxidants and free radical species that generate the signal in each assay. Cabernet Sauvignon was the red wine with the highest antioxidant capacity in comparison with Malbec and blended wine. It is concluded that the use of sensitive biological systems (as the aortic ring chemiluminescence) provides important information in addition to the results from chemical (NADH oxidation by peroxynitrite) and biochemical (homogenate chemiluminescence) assays and offers advances in the physiological role of polyphenols.