Darío E. Iglesias

Mitochondrial nitric oxide metabolism during rat heart adaptation to high altitude: effect of sildenafil, l-NAME, and l-arginine treatments

Rats submitted to high altitude (Cerro de Pasco, Perú, 4,340 m, Po(2) = 12.2 kPa) for up to 84 days showed a physiological adaptive response with decreased body weight gain (15%), increased right ventricle weight (100%), and increased hematocrit (40%) compared with sea level animals. These classical parameters of adaptation to high altitude were accompanied by an increase in heart mitochondrial enzymes: complexes I-III activity by 34% and mitochondrial nitric oxide synthase (mtNOS) activity and expression by >75%. The hyperbolic increase for mtNOS activity during adaptation to high altitude was similar to the observed pattern for hematocrit. Hematocrit and mtNOS activity mean values correlated linearly (r(2) = 0.75, P <or= 0.05). Chronic treatment for 28 days with sildenafil (50 mg*kg(-1).day(-1)) decreased the response of mtNOS to high altitude by 25%. Conversely, N(G)-nitro-l-arginine methyl ester treatment (8.3 mg*kg(-1)*day(-1)) increased such response by 40%, whereas l-arginine treatment (106 mg*kg(-1)*day(-1)) had no effect. Nitric oxide (NO) production by mtNOS accounts for approximately 49% of total cellular NO production in sea level rats and for approximately 54% in rats exposed to high altitude for 84 days. It is concluded that mtNOS is a substantial source of cardiac NO, a factor in the adaptive response to sustained heart hypoxia that is susceptible to be modified by pharmacological treatments.

Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222 ± 4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 μM GSNO and by 48% in the presence of 30 μM SPER-NO, in both cases at ~1.25 μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220 ± 9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 μM) reduced cytochrome b562 by 71%, in an [O2] independent manner. Hyperbolic increases in O2(•-) (up to 1.3 ± 0.1 nmol/min. mg protein) and H2O2 (up to 0.64 ± 0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 μM GSNO. The O2(•-)/H2O2 ratio was 1.98 in accordance with the stoichiometry of the O2(•-) disproportionation. Moreover, H2O2 production was increased by 72-74% when heart coupled mitochondria were exposed to 500 μM GSNO or 30 μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N2 atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH(•)]ss which, in turn, leads to an increase in O2(•-) and H2O2 mitochondrial production rates.

Diabetes impairs heart mitochondrial function without changes in resting cardiac performance

Diabetes is a chronic disease associated to a cardiac contractile dysfunction that is not attributable to underlying coronary artery disease or hypertension, and could be consequence of a progressive deterioration of mitochondrial function. We hypothesized that impaired mitochondrial function precedes Diabetic Cardiomyopathy. Thus, the aim of this work was to study the cardiac performance and heart mitochondrial function of diabetic rats, using an experimental model of type I Diabetes. Rats were sacrificed after 28days of Streptozotocin injection (STZ, 60mgkg-1, ip.). Heart O2 consumption was declined, mainly due to the impairment of mitochondrial O2 uptake. The mitochondrial dysfunction observed in diabetic animals included the reduction of state 3 respiration (22%), the decline of ADP/O ratio (∼15%) and the decrease of the respiratory complexes activities (22-26%). An enhancement in mitochondrial H2O2 (127%) and NO (23%) production rates and in tyrosine nitration (58%) were observed in heart of diabetic rats, with a decrease in Mn-SOD activity (∼50%). Moreover, a decrease in contractile response (38%), inotropic (37%) and lusitropic (58%) reserves were observed in diabetic rats only after a β-adrenergic stimulus. Therefore, in conditions of sustained hyperglycemia, heart mitochondrial O2 consumption and oxidative phosphorylation efficiency are decreased, and H2O2 and NO productions are increased, leading to a cardiac compromise against a work overload. This mitochondrial impairment was detected in the absence of heart hypertrophy and of resting cardiac performance changes, suggesting that mitochondrial dysfunction could precede the onset of diabetic cardiac failure, being H2O2, NO and ATP the molecules probably involved in mitochondrion-cytosol signalling.

Mitochondrial Complex I Inactivation After Ischemia-Reperfusion in the Stunned Heart

Mitochondrial complex I (NADH-ubiquinone oxidoreductase) catalyzes the transfer of two electrons from NADH via flavin mononucleotide (FMN) and a series of iron-sulfur centers (Fe-S) to ubiquinone (UQ) in a reaction associated with proton translocation across the inner membrane, contributing to the proton-motive force. Complex I produces superoxide anion (O2−) through the autoxidation reaction of the flavin-semiquinone (FMNH•) with molecular oxygen. Superoxide reacts with nitric oxide (NO) to yield peroxynitrite (ONOO−), a strong oxidant and nitrating compound. When the steady-state concentration of ONOO− is enhanced, tyrosine nitration, protein oxidation and damage to Fe-S centers take place, leading to a sustained complex I inhibition. Dysfunction of complex I was found in a number of clinical conditions such as Parkinson’s disease, ischemia-reperfusion, endotoxic shock, and aging. We have shown that the ventricular dysfunction observed in myocardial stunning is associated with a mitochondrial dysfunction that includes partial inactivation of complex I and mitochondrial nitric oxide synthase (mtNOS) activities, oxidative and nitrosative damages and increased H2O2 and ONOO− production rates. Moreover, adenosine proved to be effective in attenuating ventricular dysfunction and also in protecting from mitochondrial dysfunction and complex I syndrome.

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.

Biochemistry and Physiology of Heart Mitochondrial Nitric Oxide Synthase

Heart mitochondria are the major source of reactive oxygen and nitrogen species and play a central role in cell energy provision and signaling. The NO produced by cardiac mtNOS is allowed to interact restrictedly with the co-localized effectors. NO exerts a high affinity, reversible and physiological inhibition of cytochrome c oxidase activity. A second effect of NO on the respiratory chain is accomplished through its interaction with ubiquinol-cytochrome c oxidoreductase. The ability of mtNOS to regulate mitochondrial O2 uptake and O2− and H2O2 productions is named mtNOS functional activity. Several situations, including chronic hypoxia and ischemia-reperfusion, modify heart mtNOS activity or expression. The regulation of heart mtNOS by distinctive mitochondrial environments includes the effects of Ca2+, O2, L-arginine, NADPH, mitochondrial membrane potential (Δψ) and the metabolic states. Together, this enzyme seems to be critical during the adaptation of heart mitochondria to changes in cellular bioenergetics.

Biochemical and Morphological Alterations in Hearts of Copper-Deficient Bovines

Copper deficiency is an important disease of cattle that produces several clinical signs and lesions, due to alterations in copper-dependent enzymes. One of the organs affected by this deficiency is the heart (falling disease), but nevertheless, these cardiac lesions have not been extensively studied in bovines. The aim of this work was to propose a possible pathogenic mechanism for cardiac lesions in cattle affected by copper deficiency. Because of the possible existence of oxidative distress caused by low levels of copper-zinc-superoxide dismutase and cytochrome oxidase, ultrastructural and histological lesions have been evaluated in the heart of bovines in which a Cu deficiency had been induced using high molybdenum and sulfur levels in the diet. Our results indicated that copper deficiency produces significant damage in myocardium with high levels of lipid oxidation and a significant reduction in copper-zinc-superoxide dismutase activity leading to an oxidative distress situation. However, cytochrome oxidase activity was not significantly reduced. Histological observation revealed a significant increase in the amount of connective tissue, enlarged basement membranes of myocytes, and numerous Anichkov cells, in the hearts of deficient animals. Ultrastructural observation showed a significant enhancement in the mitochondrial volume density, with presence of lesions such as swelling and cristae disruption. We conclude that copper deficiency in bovines causes morphological lesions in the heart due to an oxidative damage produced by copper-dependent enzyme alterations.