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.

Effects of Apocynin on Heart Muscle Oxidative Stress of Rats with Experimental Diabetes: Implications for Mitochondria.

Diabetes mellitus (DM) constitutes one of the public health problems today. It is characterized by hyperglycemia through a defect in the ß-cells function and/or decreased insulin sensitivity. Apocynin has been tasted acting directly as an NADPH oxidase inhibitor and reactive oxygen species (ROS) scavenger, exhibiting beneficial effects against diabetic complications. Hence, the present study's goal was to dissect the possible mechanisms by which apocynin could mediate its cardioprotective effect against DM-induced oxidative stress. Male Wistar rats were assigned into 4 groups: Control (C), control + apocynin (C+A), diabetes (D), diabetes + apocynin (D+A). DM was induced with streptozotocin. Apocynin treatment (3 mg/kg/day) was applied for 5 weeks. Treatment significantly decreased blood glucose levels and insulin resistance in diabetic rats. In cardiac tissue, ROS levels were higher, and catalase enzyme activity was reduced in the D group compared to the C group; the apocynin treatment significantly attenuated these responses. In heart mitochondria, Complexes I and II of the electron transport chain (ETC) were significantly enhanced in the D+A group. Total glutathione, the level of reduced glutathione (GSH) and the GSH/ oxidized glutathione (GSSG) ratio were increased in the D+A group. Superoxide dismutase (SOD) and the glutathione peroxidase (GSH-Px) activities were without change. Apocynin enhances glucose uptake and insulin sensitivity, preserving the antioxidant defense and mitochondrial function.

Association between ROS production, swelling and the respirasome integrity in cardiac mitochondria.

Although mitochondrial Ca2+ overload and ROS production play a critical role in mitochondria-mediated cell death, a cause-effect relationship between them remains elusive. This study elucidated the crosstalk between mitochondrial swelling, ROS production, and electron transfer chain (ETC) supercomplexes in rat heart mitochondria in response to Ca2+ and tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide. Results showed that ROS production induced by TBH was significantly increased in the presence of Ca2+ in a dose-dependent manner. TBH markedly inhibited the state 3 respiration rate with no effect on the mitochondrial swelling. Ca2+ exerted a slight effect on mitochondrial respiration that was greatly aggravated by TBH. Analysis of supercomplexes revealed a minor difference in the presence of TBH and/or Ca2+. However, incubation of mitochondria in the presence of high Ca2+ (1 mM) or inhibitors of ETC complexes (rotenone and antimycin A) induced disintegration of the main supercomplex, respirasome. Thus, PTP-dependent swelling of mitochondria solely depends on Ca2+ but not ROS. TBH has no effect on the respirasome while Ca2+ induces disintegration of the supercomplex only at a high concentration. Intactness of individual ETC complexes I and III is important for maintenance of the structural integrity of the respirasome.

Hydrogen peroxide, nitric oxide and ATP are molecules involved in cardiac mitochondrial biogenesis in Diabetes.

This study, in an experimental model of type I Diabetes Mellitus in rats, deals with the mitochondrial production rates and steady-state concentrations of H2O2 and NO, and ATP levels as part of a network of signaling molecules involved in heart mitochondrial biogenesis. Sustained hyperglycemia leads to a cardiac compromise against a work overload, in the absence of changes in resting cardiac performance and of heart hypertrophy. Diabetes was induced in male Wistar rats by a single dose of Streptozotocin (STZ, 60mg × kg-1, ip.). After 28 days of STZ-injection, rats were sacrificed and hearts were isolated. The mitochondrial mass (mg mitochondrial protein × g heart-1), determined through cytochrome oxidase activity ratio, was 47% higher in heart from diabetic than from control animals. Stereological analysis of cardiac tissue microphotographs showed an increase in the cytosolic volume occupied by mitochondria (30%) and in the number of mitochondria per unit area (52%), and a decrease in the mean area of each mitochondrion (23%) in diabetic respect to control rats. Additionally, an enhancement (76%) in PGC-1α expression was observed in cardiac tissue of diabetic animals. Moreover, heart mitochondrial H2O2 (127%) and NO (23%) productions and mtNOS expression (132%) were higher, while mitochondrial ATP production rate was lower (~ 40%), concomitantly with a partial-mitochondrial depolarization, in diabetic than in control rats. Changes in mitochondrial H2O2 and NO steady-state concentrations and an imbalance between cellular energy demand and mitochondrial energy transduction could be involved in the signaling pathways that lead to the novo synthesis of mitochondria. However, this compensatory mechanism triggered to restore the mitochondrial and tissue normal activities, did not lead to competent mitochondria capable of supplying the energetic demands in diabetic pathological conditions.

In female rat heart mitochondria, oophorectomy results in loss of oxidative phosphorylation.

Oophorectomy in adult rats affected cardiac mitochondrial function. Progression of mitochondrial alterations was assessed at one, two and three months after surgery: at one month, very slight changes were observed, which increased at two and three months. Gradual effects included decrease in the rates of oxygen consumption and in respiratory uncoupling in the presence of complex I substrates, as well as compromised Ca2+ buffering ability. Malondialdehyde concentration increased, whereas the ROS-detoxifying enzyme Mn2+ superoxide dismutase (MnSOD) and aconitase lost activity. In the mitochondrial respiratory chain, the concentration and activity of complex I and complex IV decreased. Among other mitochondrial enzymes and transporters, adenine nucleotide carrier and glutaminase decreased. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase also decreased. Data strongly suggest that in the female rat heart, estrogen depletion leads to progressive, severe mitochondrial dysfunction.