Running head: Chronic hypoxia and heart mtNOS Postal address

Frontiers in Bioscience, 2007

Journal of Molecular and Cellular Cardiology, 2007

The objective of the present study was to delineate the molecular mechanisms for mitochondrial contribution to oxidative stress induced by hypoxia and reoxygenation in the heart. The present study introduces a novel model allowing real-time studying mitochondria under hypoxia and reoxygenation, and describes the significance of intramitochondrial calcium homeostasis and mitochondrial nitric oxide synthase (mtNOS) for oxidative stress. The present study shows that incubating isolated rat heart mitochondria under hypoxia followed by reoxygenation, but not hypoxia per se, causes cytochrome c release from the mitochondria, oxidative modification of mitochondrial lipids and proteins, and inactivation of mitochondrial enzymes susceptible to inactivation by peroxynitrite. Those alterations were prevented when mtNOS was inhibited or mitochondria were supplemented with antioxidant peroxynitrite scavengers. The present study shows mitochondria independent of other cellular components respond to hypoxia/reoxygenation by elevating intramitochondrial ionized calcium and stimulating mtNOS. The present study proposes a crucial role for heart mitochondrial calcium homeostasis and mtNOS in oxidative stress induced by hypoxia/ reoxygenation.

Abstract: Chronic hypoxia alters mitochondrial energy metabolism. In the heart, oxidative capacity of both ventricles is decreased after 3 weeks of chronic hypoxia. The aim of this study was to evaluate the reversal of these metabolic changes upon normoxia recovery. Rats were exposed to a hypobaric environment for 3 weeks and then subjected to a normoxic environment for 3 weeks (normoxia-recovery group) and compared with rats maintained in a normoxic environment (control group). Mitochondrial energy metabolism was differentially examined in both left and right ventricles. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers. Activities of mitochondrial respiratory chain complexes and antioxidant enzymes were measured on ventricle homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. In normoxia-recovery rats, oxidative capacities of right ventricles were decreased in the presence of glutamate or palmitoyl carnitine as substrates. In contrast, oxidation of palmitoyl carnitine was maintained in the left ventricle. Enzyme activities of complexes III and IV were significantly decreased in both ventricles. These functional alterations were associated with a decrease in numerical density and an increase in size of mitochondria. Finally, in the normoxia-recovery group, the antioxidant enzyme activities (catalase and glutathione peroxidase) increased. In conclusion, alterations of mitochondrial energy metabolism induced by chronic hypoxia are not totally reversible. Reactive oxygen species could be involved and should be investigated under such conditions, since they may represent a therapeutic target.

Journal of Biological Chemistry, 2003

Nitric oxide (NO ⅐) inhibits mitochondrial respiration by binding to the binuclear heme a 3 /Cu B center in cytochrome c oxidase. However, the significance of this reaction at physiological O 2 levels (5-10 M) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O 2 ], and [NO ⅐ ] were measured simultaneously at physiological O 2 levels with constant O 2 delivery, to model in vivo respiratory dynamics. Under these conditions NO ⅐ inhibited mitochondrial respiration with an IC 50 of 0.14 ؎ 0.01 M in state 3 versus 0.31 ؎ 0.04 M in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO ⅐ is due to greater control over respiration by an NO ⅐-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO ⅐ in vivo and its implications for the control of vessel-parenchymal O 2 gradients.

Circulation, 1999

Background-Our objective for this study was to investigate whether nitric oxide (NO) modulates tissue respiration in the failing human myocardium. Methods and Results-Left ventricular free wall and right ventricular tissue samples were taken from 14 failing explanted human hearts at the time of transplantation. Tissue oxygen consumption was measured with a Clark-type oxygen electrode in an airtight stirred bath containing Krebs solution buffered with HEPES at 37 degrees C (pH 7.4). Rate of decrease in oxygen concentration was expressed as a percentage of the baseline, and results of the highest dose are indicated. Bradykinin (10(-4) mol/L, -21+/-5%), amlodipine (10(-5) mol/L, -14+/-5%), the ACE inhibitor ramiprilat (10(-4) mol/L, -21+/-2%), and the neutral endopeptidase inhibitor thiorphan (10(-4) mol/L, -16+/-5%) all caused concentration-dependent decreases in tissue oxygen consumption. Responses to bradykinin (-2+/-6%), amlodipine (-2+/-4%), ramiprilat (-5+/-6%), and thiorphan (-4+/-7%) were significantly attenuated after NO synthase blockade with N-nitro-L-arginine methyl ester (10(-4) mol/L; all P<0.05). NO-releasing compounds S-nitroso-N-acetyl-penicillamine (10(-4) mol/L, -34+/-5%) and nitroglycerin (10(-4) mol/L, -21+/-5%), also decreased tissue oxygen consumption in a concentration-dependent manner. However, the reduction in tissue oxygen consumption in response to S-nitroso-N-acetyl-penicillamine (-35+/-7%) or nitroglycerin (-16+/-5%) was not significantly affected by N-nitro-L-arginine methyl ester. Conclusions-These results indicate that the modulation of oxygen consumption by both endogenous and exogenous NO is preserved in the failing human myocardium and that the inhibition of kinin degradation plays an important role in the regulation of mitochondrial respiration.

Frontiers in Bioscience-Scholar, 2021

Introduction 3. Energy-producing function of mitochondria and H 2 S 4. The role of nitric oxide and mitochondrial nitric oxide synthase in cardioprotection during hypoxia and ischemia/reperfusion 5. The role of carbon monoxide and heme oxygenase system in vasodilation. Antiarrhythmic effects of endogenic CO 6. Antiapoptotic properties of CO, H 2 S and NO 6.1 Antiapoptotic properties of CO 6.2 Antiapoptotic properties of H 2 S 6.3 Antiapoptotic properties of NO 7. Interactions of H 2 S, NO and CO in the cardiovascular system during hypoxia 8.

European journal of applied physiology, 2018

Exposure to hypoxia has been suggested to activate multiple adaptive pathways so that muscles are better able to maintain cellular energy homeostasis. However, there is limited research regarding the tissue specificity of this response. The aim of this study was to investigate the influence of tissue specificity on mitochondrial adaptations of rat skeletal and heart muscles after 4 weeks of normobaric hypoxia (FiO: 0.10). Twenty male Wistar rats were randomly assigned to either normobaric hypoxia or normoxia. Mitochondrial respiration was determined in permeabilised muscle fibres from left and right ventricles, soleus and extensorum digitorum longus (EDL). Citrate synthase activity and the relative abundance of proteins associated with mitochondrial biogenesis were also analysed. After hypoxia exposure, only the soleus and left ventricle (both predominantly oxidative) presented a greater maximal mass-specific respiration (48 and 25%, p < 0.05) and mitochondrial-specific respirati...

Basic Research in Cardiology, 2004

Recent publications shown mitochondrial localization of the enzyme nitric oxide synthase (NOS) in a number of tissues. However, conflicting results about mitochondrial NOS (mtNOS) immunoreactivity and enzymatic activity are available to date in the literature. In this study we purified mitochondria from rat hearts and analysed these preparations for NOS immunoreactivity and activity, showing the presence of either a constitutive (the endothelial isoform) and an inducible NOS immunoreactivity. A basal NOS activity (64.2 ± 5.1 pmol/mg protein/30 min) was detectable. 1 mM N G -Monomethyl-L-arginine (L-NMMA), a competitive inhibitor of all NOS isoforms, caused a drop in NOS activity to 33.8 ± 1.9 pmol/mg protein/30 min. Simultaneous administration of 10 µM (S)-2-amino-(1-iminoethylamino)-5thiopentanoic acid (GW274150), a specific NOS2 inhibitor, together with removal of Ca 2+ and calmodulin (CaM) from the assay buffers, known to interfere with the activity of constitutive NOS isoforms, caused a reduction in NOS activity (17.4 ± 1.2 pmol/mg protein/30 min). 10 µM GW274150 reduced NOS activity to 41.6 ± 4 pmol/mg protein/30 min, while Ca 2+ /CaM withdrawal reduced basal NOS activity to 45.8 ± 5 pmol/mg protein/30 min. This dual mtNOS machinery is suggested to be involved in modulating cardiac O 2 consumption in different (patho)physiological conditions. Fig. 3 Modulation of NOS activity in Percoll purified rat heart mitochondria. [ 3 H]Larginine conversion to [ 3 H]L-citrulline. Values are mean Ȁ SEM of five separate experiments. Statistical analysis was performed using [one-way analysis of variance (ANOVA): p < 0.01)] and [Dunnett post-test: * = p < 0.01 vs. Control]

Life Sciences, 2007

Previous studies raised the possibility that nitric oxide synthase is present in heart mitochondria (mtNOS) and the existence of such an enzyme became generally accepted. However, original experimental evidence is rather scarce and positive identification of the enzyme is lacking. We aimed to detect an NOS protein in human and mouse heart mitochondria and to measure the level of NO released from the organelles. Western blotting with 7 different anti-NOS antibodies failed to detect a NOS-like protein in mitochondria. Immunoprecipitation or substrate-affinity purification of the samples concentrated NOS in control preparations but not in mitochondria. Release of NO from live respiring human mitochondria was below 2 ppb after 45 min of incubation. In a bioassay system, mitochondrial suspension failed to cause vasodilation of human mammary artery segments. These results indicate that mitochondria do not produce physiologically relevant quantities of NO in the heart and are unlikely to have any physiological importance as NO donors, nor do they contain a recognizable mtNOS enzyme.

Journal of Molecular and Cellular Cardiology, 1999

Induction of nitric oxide synthase (NOS2, also designated as iNOS) in the heart is known to occur in response to various stimuli. It is not known, however, whether in vivo hypoxia leads to cardiac NOS2 induction. We thus investigated the effects of normobaric hypoxia (10% O 2 for 8, 15 and 21 days) on NOS2 protein expression and enzyme activity in rat right ventricle (RV) and left ventricle (LV). Chronic hypoxia induced RV hypertrophy: the RV weight to body weight ratio was increased by 45% upon 15 days of exposure, with no change thereafter and no change in left ventricular (LV) weight. Treatment of hypoxic rats with -NAME for 1 month decreased pulmonary artery pressure and RV hypertrophy compared to hypoxic non-treated rats. NOS2 activity detected by [ 3 H] -arginine to [ 3 H] -citrulline conversion increased in RV during hypoxia, with a maximum at 15 days (+161% of control rats; P<0.05), whereas it increased less (by 60%) in LV. In parallel, after 15 days of hypoxia there was a three-fold increase in NOS2 protein abundance detected by Western blotting using an isoform-specific antibody in the RVs (two-fold increase in the LV). Immunochemistry with the specific antibody demonstrated the expression in cardiomyocytes isolated from both ventricles of normoxic and hypoxic rats. Protein kinase C (PKC) content and activity was unchanged in LV of hypoxic rats, but increased in RV as compared with normoxic rats. These results clearly show that, in the heart, NOS2 is upregulated by hypoxia with an expression in cardiomyocytes of both ventricles. In addition, NOS2 is more inducible in the right hypertrophied ventricle than in the left non-hypertrophied hypoxic ventricle.

American Journal of Hypertension, 2008

Nitric oxide (NO) is involved in the control of cardiovascular function in physiological conditions and in heart diseases. As the prototypical endothelium-derived relaxing factor, NO is a primary determinant of blood vessel tone and thrombogenicity. In the context of heart tissue, these functions themselves are sufficient to justify the growing interest in NO as a regulator of cardiac function. However, despite the extensive literature, the exact sites and nature of NO action in the heart are as yet uncertain, while the earlier hypothesis that all three genomic forms of NO synthase (NOS) are active in the heart, have given rise to several intriguing questions. The classic concepts linking heart function with NO are: (i) NO is a regulator of cardiac function through direct action on the myocardium, and also by indirect vascular-dependent mechanisms 1 and (ii) the three known genomic isoforms of NOS 2 are present and functionally active in the heart. Gonzales et al. 3 and Zaobornyj et al. 4 challenged the second concept and reported that NO is produced in physiological conditions in the myocardium in relevant quantities by two of the isoforms of NOS: (i) an isoenzyme located in the mitochondria, known as mitochondrial NOS (mtNOS) and (ii) an isoenzyme located in the cytosolic fraction, the endothelial NOS (eNOS). The mechanisms by which NO regulates heart contractility and contraction rate, and the relation between the heart cycle and diffusion of NO between mitochondria and cytosol are physiological processes that are only now beginning to be understood. In this paper we attempt to revisit the major concepts about the influence of NO on heart rate, with special focus on the role of mitochondrial NO.

European Journal of Histochemistry, 2011

Hypoxia/reoxygenation (H/R) reportedly influences nitric oxide (NO) production and NO synthase (NOS) expression in the heart. Nonetheless, a number of works have shown controversial results regarding the changes that the cardiac NO/NOS system undergoes under such situations. Therefore, this study aims to clarify the behaviour of this system in the hypoxic heart by investigating seven different reoxygenation times. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0, 2, 12, 24, 48, 72 h, and 5 days) in a novel approach to address the events provoked by assaults under such circumstances. Endothelial and inducible NOS (eNOS and iNOS) mRNA and protein expression, as well as enzymatic activity and enzyme location were determined. NO levels were indirectly quantified as nitrate/nitrite, and other Snitroso compounds (NOx), which would act as NO-storage molecules. The results showed a significant increase in eNOS mRNA, protein and activity, as well as in NOx levels immediately after hypoxia, while iNOS protein and activity were induced throughout the reoxygenation period. These findings indicate that, not only short-term hypoxia, but also the subsequent reoxygenation period upregulate cardiac NO/NOS system until at least 5 days after the hypoxic stimulus, implying major involvement of this system in the changes occurring in the heart in response to H/R.

American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2022

Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular disease that is characterized by chronic intermittent hypoxia (CIH), and its impact is related to age. This study aims to assess the age-related impact of CIH on cardiac function and to further explore the mechanism. After 8 wk of severe CIH exposure, the hearts of young mice showed slight physiological hypertrophy, decreased diastolic function, and collagen I accumulation but no obvious change in contractile function. However, the contractile function of the hearts of aged mice was severely decreased. CIH exposure promoted the fragmentation of mitochondria in the hearts of aged mice and decreased the mitochondrial membrane potential of cardiomyocytes, but these effects were not observed in young mice exposed to the same conditions. CIH induced significant decreases in basal respiration, maximum respiration, and ATP production in cardiac mitochondria of aged mice compared with those of young mice. The ass...

Frontiers in Bioscience, 2007

Mitochondria produce nitric oxide (NO) through a Ca 2Csensitive mitochondrial NO synthase (mtNOS). The NO produced by mtNOS regulates mitochondrial oxygen consumption and transmembrane potential via a reversible reaction with cytochrome c oxidase. The reaction of this NO with superoxide anion yields peroxynitrite, which irreversibly modifies susceptible targets within mitochondria and induces oxidative and/or nitrative stress. In this article, we review the current understanding of the roles of mtNOS as a crucial biochemical regulator of mitochondrial functions and attempt to reconcile apparent discrepancies in the literature on mtNOS. Discovery of mitochondrial nitric oxide synthase The discovery that the endothelium-derived relaxing factor is nitric oxide (NO) [1] opened new horizons in biomedical research. The cellular synthesis of NO is catalyzed by NO synthase (NOS) isozymes, three of which are well characterized. Although expression of these enzymes is not tissue specific, they are referred to as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Each isozyme consumes L-arginine, produces equal amounts of NO and L-citrulline, and requires Ca 2C-calmodulin for activity. The activity of eNOS and nNOS are regulated tightly by alterations in Ca 2C status but, because iNOS forms a complex with calmodulin at very low concentrations of Ca 2C , its activity is not regulated by Ca 2C alterations. NO exerts a broad spectrum of functions in several system, including the cardiovascular system, PNS, CNS and immune system. These functions are mediated through the reactions of NO with targets that include hemoproteins, thiols and superoxide anions. Mitochondria possess several hemoproteins (e.g. cytochrome c oxidase), thiols (e.g. glutathione) and cysteine-containing proteins, and they are major cellular sources of superoxide anion. Consequently, mitochondria are important targets of NO and contribute to several of the biological functions of NO [2]. Several laboratories have addressed the possibility that NOS is present in mitochondria. The cross-reaction of mitochondria with antibodies to Ca 2C-sensitive eNOS was reported almost simultaneously by two laboratories. In rats, mitochondria from skeletal muscle fibers from the diaphragm [3], non-synaptosomal brain [4], and heart, skeletal muscle and kidney [5] cross-react with eNOS antibodies. Other laboratories also report an association

Nitric Oxide, 2018

In previous studies, upregulation of NOS during acclimatization of rats to sustained hypobaric hypoxia was associated to cardioprotection, evaluated as an increased tolerance of myocardium to hypoxia/reoxygenation. The objective of the present work was to investigate the effect of acute hypobaric hypoxia and the role of endogenous NO concerning cardiac tolerance to hypoxia/reoxygenation under β-adrenergic stimulation. Methods: Rats were submitted to 58.7 kPa in a hypopressure chamber for 48 h whereas their normoxic controls remained at 101.3 kPa. By adding NOS substrate L-arg, or blocker L-NNA, isometric mechanical activity of papillary muscles isolated from left ventricle was evaluated at maximal or minimal production of NO, respectively, under β-adrenergic stimulation by isoproterenol, followed by 60/30 min of hypoxia/reoxygenation. Activities of NOS and cytochrome oxidase were evaluated by spectrophotometric methods and expression of HIF1-α and NOS isoforms by western blot. Eosin and hematoxiline staining were used for histological studies. Results: Cytosolic expression of HIF1-α, nNOS and eNOS, and NO production were higher in left ventricle of hypoxic rats. Mitochondrial cytochrome oxidase activity was decreased by hypobaric hypoxia and this effect was reversed by L-NNA. After H/R, recovery of developed tension in papillary muscles from normoxic rats was 51-60% (regardless NO modulation) while in hypobaric hypoxia was 70% ± 3 (L-arg) and 54% ± 1 (L-NNA). Other mechanical parameters showed similar results. Preserved histological architecture was observed only in Larg papillary muscles of hypoxic rats. Conclusion: Exposure of rats to hypobaric hypoxia for only 2 days increased NO synthesis leading to cardioprotection.