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National Heart, Lung, and Blood Institute UAB AMC21 reload multi-investigator grant At various biological levels, mammals must integrate with 24-hr rhythms in their environment. Daily fluctuations in stimuli/stressors of cardiac metabolism and oxidation-reduction (redox) status have been reported over the course of the day. It is therefore not surprising that the heart exhibits dramatic oscillations in various cellular processes over the course of the day, including transcription, translation, ion homeostasis, metabolism, and redox signaling. This temporal partitioning of cardiac processes is governed by a complex interplay between intracellular (e.g., circadian clocks) and extracellular (e.g., neurohumoral factors) influences, thus ensuring appropriate responses to daily stimuli/ stresses. The purpose of the current article is to review knowledge regarding control of metabolism and redox biology in the heart over the course of the day, and to highlight whether disruption of these daily rhythms contribute towards cardiac dysfunction observed in various disease states. Univ Fed Sao Paulo, Dept Biol Sci, Morphophysiol & Pathol Sect, Diadema, SP, Brazil Univ Alabama Birmingham, Dept Pathol, Mitochondrial Med Lab, Birmingham, AL 35294 USA Univ Alabama Birmingham, Dept Med, Div Cardiovasc Dis, 703 19th St S,ZRB 308, Birmingham, AL 35294 USA Univ Fed Sao Paulo, Dept Biol Sci, Morphophysiol & Pathol Sect, Diadema, SP, Brazil NHLBI: HL106199 NHLBI: HL074259 NHLBI: HL123574 NHLBI: HL122975 Web of Science

Platelet aggregation is an essential response to tissue injury and is associated with activation of pro-oxidant enzymes, such as cyclooxygenase, and is also a highly energetic process. The two central energetic pathways in the cell, glycolysis and mitochondrial oxidative phosphorylation, are susceptible to damage by reactive lipid species. Interestingly, how platelet metabolism is affected by the oxidative stress associated with aggregation is largely unexplored. To address this issue, we examined the response of human platelets to 4-hydroxynonenal (4-HNE), a reactive lipid species which is generated during thrombus formation and during oxidative stress. Elevated plasma 4-HNE has been associated with renal failure, septic shock and cardiopulmonary bypass surgery. In this study, we found that 4-HNE decreased thrombin stimulated platelet aggregation by approximately 60%. The metabolomics analysis demonstrated that underlying our previous observation of a stimulation of platelet energetics by thrombin glycolysis and TCA (Tricarboxylic acid) metabolites were increased. Next, we assessed the effect of both 4-HNE and alkyne HNE (A-HNE) on bioenergetics and targeted metabolomics, and found a stimulatory effect on glycolysis, associated with inhibition of bioenergetic parameters. In the presence of HNE and thrombin glycolysis was further stimulated but the levels of the TCA metabolites were markedly suppressed. Identification of proteins modified by A-HNE followed by click chemistry and mass spectrometry revealed essential targets in platelet activation including proteins involved in metabolism, adhesion, cytoskeletal reorganization, aggregation, vesicular transport, protein folding, antioxidant proteins, and small GTPases. In summary, the biological effects of 4-HNE can be more effectively explained in platelets by the integrated effects of the modification of an electrophile responsive proteome rather than the isolated effects of candidate proteins.

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species (ROS/RNS) under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons maintained the same basal cellular respiration in hypoxia as normoxia, whereas NO exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO treated cells did not recover to pre-hypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy and the inability to recovery during reoxygenation was exacerbated by the inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the media or glycolysis was inhibited by 2-deoxy-D-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration which is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into understanding how autophagy and NO may play an interactive role in neuroinflammation-induced cellular damage which is pertinent to our understanding of the pathology neurodegenerative diseases in which excessive NO is generated.

An important emerging area in the translational aspects of redox biology and bioenergetics are the new findings that implicate mitochondrial dysfunction as a key player in the etiology of metabolic syndrome and diabetes. The paper in this issue of Free Radical Biology & Medicine, entitled “The mitochondria-targeted antioxidant MitoQ decreases features of the Metabolic Syndrome in ATM+/−/ApoE−/− mice” by Mercer and colleagues shows, in an elegant pre-clinical study, that mitochondrial therapeutics may be beneficial for this important clinical problem. The work is exciting because it not only validates a role for mitochondrial redox signaling in the etiology of metabolic syndrome but also highlights a potential therapeutic agent to treat metabolic syndrome in human subjects.

The endothelium is not considered to be a major energy-requiring organ, but nevertheless endothelial cells have an extensive mitochondrial network. This suggests that mitochondrial function may be important in response to stress and signaling in these cells. In this study, we used extracellular flux analysis to measure mitochondrial function in adherent bovine aortic endothelial cells (BAEC). Under basal conditions, BAEC use only approximately 35% of their maximal respiratory capacity. We calculate that this represents an intermediate respiratory state between States 3 and 4, which we define as State(apparent) equal to 3.64. Interestingly, the apparent respiratory control ratio (maximal mitochondrial oxygen consumption/non-ADP-linked respiration) in these cells is on the order of 23, which is substantially higher than that which is frequently obtained with isolated mitochondria. These results suggest that mitochondria in endothelial cells are highly coupled and possess a considerable bioenergetic reserve. Because endothelial cells are exposed to both reactive oxygen (ROS) and reactive nitrogen species in the course of vascular disease, we hypothesized that this reserve capacity is important in responding to oxidative stress. To test this, we exposed BAEC to NO or ROS alone or in combination. We found that exposure to nontoxic concentrations of NO or low levels of hydrogen peroxide generated from 2,3-dimethoxy-1,4-napthoquinone (DMNQ) had little impact on basal mitochondrial function but both treatments reversibly decreased mitochondrial reserve capacity. However, combined NO and DMNQ treatment resulted in an irreversible loss of reserve capacity and was associated with cell death. These data are consistent with a critical role for the mitochondrial reserve capacity in endothelial cells in responding to oxidative stress.

The combination of proteomics with highly specific and sensitive affinity techniques is important for the identification of posttranslational modifications by reactive oxygen and nitrogen species (ROS/RNS). One of the most pressing problems with this approach is to determine accurately the extent of modification of specific amino acids, such as cysteine residues, in a complex protein sample. A number of techniques relevant to free radical biology use biotin tagging as a method to follow protein modification with high sensitivity and specificity. To realize the potential of this approach to provide quantitative data, we have prepared a series of biotinylated proteins through the modification of lysine residues. These proteins were then used as quantitative standards in electrophoretic separation of protein samples labeled with biotin-conjugated iodoacetamide. The utility of the approach was assessed by measuring modification of thiols in response to exposure to thiol oxidants, as well as the amount of protein adduct formation with a biotin-tagged electrophilic lipid. Furthermore, using a combination of native and biotin-tagged cytochrome c, this method was used to quantitate the amount of thiol relative to the amount of protein in a given spot on a two-dimensional gel. Thus, we have developed a versatile, cost-effective standard that can be used in proteomic methods to quantitate biotin tags in response to oxidative stress.

Epidemiological evidence indicates that moderate alcohol consumption reduces the incidence of heart disease. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis and myocardial functions through the controlled production of nitric oxide (*NO). These studies were conducted to determine if the apparent alcohol-associated cardioprotection is mediated, in part, through modulation of the eNOS protein and activity in the cardiovascular system. Rats were fed alcohol and eNOS protein and *NO production were evaluated at the end of 8 weeks. Myocardial and vascular function was assessed ex vivo in a subset of animals. Moderate alcohol improved postischemic myocardial systolic and diastolic function and attenuated the postischemic reduction in coronary vascular resistance. Moderate alcohol also enhanced maximum vascular relaxation by 26 +/- 0.2% and increased plasma *NO production concomitant with a greater than 2.5-fold increase in eNOS protein. Higher levels of alcohol impaired maximum vascular relaxation by 22 +/- 0.1%. These results suggest that moderate alcohol improves postischemic myocardial functions and increases *NO production by vascular endothelium. An increase in *NO may explain, at least in part, the cardioprotective benefits of moderate alcohol consumption.

Cardiovascular diseases encompass a wide spectrum of abnormalities with diverse etiologies. The molecular mechanisms underlying these disorders include a variety of responses such as changes in nitric oxide- (NO) dependent cell signaling and increased apoptosis. An interesting aspect that has received little or no attention is the role mitochondria may play in the vascular changes that occur in both atherosclerosis and hypertension. With the changing perspective of the organelle from simply a role in metabolism to a contributor to signal transduction pathways, the role of mitochondria in cells with relatively low energy demands such as the endothelium has become important to understand. In this context, the definition of the NO-cytochrome c oxidase signaling pathway and the influence this has on cytochrome c release is particularly important in understanding apoptotic mechanisms involving the mitochondrion. This review examines the role of compromised mitochondrial function in a variety of vascular pathologies and the modulation of these effects by NO. The interaction of NO with the various mitochondrial respiratory complexes and the role NO plays in modulating mitochondrial-mediated apoptosis in these systems will be discussed.

Nitric oxide (NO • ) signaling is diverse, and involves reaction with free radicals, metalloproteins, and specific protein amino acid residues. Prominent among these interactions are the heme protein soluble guanylate cyclase and cysteine residues within several proteins such as caspases, the executors of apoptosis. Another well characterized site of NO • binding is the terminal complex of the mitochondrial respiratory chain, cytochrome c oxidase, although the downstream signaling effects of this interaction remain unclear. Recently, it has been recognized that the intracellular formation of hydrogen peroxide (H 2 O 2 ) by controlled mechanisms contributes to what we term “redox tone,” and so controls the activity and activation thresholds of redox-sensitive signaling pathways. In this hypothesis paper, it is proposed that NO • -dependent modulation of the respiratory chain can control the mitochondrial generation of H 2 O 2 for cell signaling purposes without affecting ATP synthesis.

S-Nitrosothiols are potentially important mediators of biological processes including vascular function, apoptosis, and thrombosis. Recent studies indicate that the concentrations of S-nitrosothiols in the plasma from healthy individuals are lower than previously reported and in the range of 30-120 nM. The mechanisms of formation and metabolism of these low nM concentrations, capable of exerting biological effects, remain unknown. An important issue that remains unresolved is the significance of the reactions of low fluxes of nitric oxide (NO) with oxygen to form S-nitrosothiols in a complex biological medium such as plasma, and the impact of red blood cells on the formation of S-nitrosothiols in blood. These issues were addressed by exposing plasma to varying fluxes of NO and measuring the net formation of S-nitrosothiols. In the presence of oxygen and physiological fluxes of NO, the predominant S-nitrosothiol formed is S-nitroso-albumin at concentrations in the high nM range (approximately 400-1000 nM). Although the formation of S-nitrosothiols by NO was attenuated in whole blood, presumably by erythrocytic hemoglobin, significant amounts of S-nitrosothiols within the physiological range of S-nitrosothiol concentrations (approximately 80 nM) were still formed at physiological fluxes of NO. Little is known about the stability of S-nitroso-albumin in plasma, and this is central to our understanding of the biological effectiveness of S-nitrosothiols. Low molecular weight thiols decreased the half-life of S-nitroso-albumin in plasma, and the stability of S-nitroso-albumin is enhanced by the alkylation of free thiols. Our data suggests that physiologically relevant concentrations of S-nitrosothiols can be formed in blood through the reaction of NO with oxygen and proteins, despite the low rates of reaction of oxygen with NO and the presence of erythrocytes.

Much of the damaging action of nitric oxide in heart may be due to its diffusion-limited reaction with superoxide to form peroxynitrite. Direct infusion of peroxynitrite into isolated perfused hearts fails to model the effects of in situ formation because the bulk of peroxynitrite decomposes before reaching the myocytes. To examine the direct effects of peroxynitrite on the contractile apparatus of the heart, we exposed intact and skinned rat papillary muscles to a steady state concentration of 4-μM peroxynitrite for 5 min, followed by a 30-min recovery period to monitor irreversible effects. In intact muscles developed force fell immediately to 26% of initial force, recovering to 43% by 30 min. Resting tension increased by 600% immediately, and was still elevated 500% by 30 min. Nitrotyrosine immunochemistry showed that peroxynitrite can induce tyrosine nitration at low concentrations and is capable of penetrating 200–380 μm into the papillary muscle after a 5-min infusion. Decomposed peroxynitrite had no effect on either intact or skinned muscle developed force or resting tension. Our results show that peroxynitrite directly damages both developed force and resting tension of isolated heart muscle, which can be extrapolated to systolic and diastolic injury in intact hearts.