These data demonstrate that nanomole doses of nitrite mediate both acute cytoprotection and mimic delayed preconditioning in the heart and liver in vivo. Mechanistically, both acute and delayed nitrite-dependent cytoprotection occur at the mitochondrial level, with the inhibition of mitochondrial complex I by S-nitrosation limiting mitochondrial ROS generation, oxidative protein damage to the electron transport chain, cytochrome c release, and cellular and tissue infarction.
Interestingly, NO production has been shown to evoke acute (classical) preconditioning and is required in the delayed ischemic preconditioning cell-survival program (19
). This occurs through increased endothelial NOS activity after acute ischemic preconditioning and via induction of iNOS 24 h later for remote preconditioning (50
). Relevant to the current study, nitrite levels increase in a biphasic manner after both acute and delayed ischemic preconditioning, with maximal increases observed during the I/R tolerant periods (50
). In this study, we show that administration of nitrite at either of these preconditioning time points confers potent cytoprotection in vivo and modulates mitochondrial function. In addition, previous studies have shown complex I inhibition (51
), as well as an increase in mitochondrial S-nitrosation (39
), after ischemic preconditioning of the rat heart. Given these data, it is intriguing to consider that nitrite could serve as both an endogenous reservoir for NO production and an effector molecule mediating the cell-survival effects of both acute and delayed ischemic preconditioning. Consistent with such NO-dependent signaling, PTIO, a direct NO scavenger, has been observed to inhibit the cytoprotective effects of nitrite in cardiac (10
), liver (20
), and brain (9
NO itself has been shown to directly protect cultured rat neonatal cardiomyocyte mitochondria when administered in time periods compatible with both classical and delayed preconditioning (52
). Previous studies show that NO modulates mitochondrial Ca2+
handling that in turn diminishes reoxygenation-associated Ca2+
). In this manuscript, we demonstrate that nitrite administration directly modifies the electron transfer chain complex I activity in association with S-nitrosation of this complex. This is associated with blunted reperfusion/reoxygenation ROS-mediated injury. The putative mechanisms that could confer this phenotype include the direct inhibition of complex I–generated ROS or via the modest reduction in the mitochondrial membrane potential that may result from the partial inhibition of complex I activity (5
An apparent paradox is evident with respect to complex I inhibition and ROS generation. In brief, in an aerobic environment with adequate oxidative phosphorylation, substrate, and reducing equivalents, the direct inhibition of complex I with either rotenone (54
) or by preformed SNOs (40
) results in complex I–derived ROS production. Conversely, when complex I is inhibited in the context of I/R by rotenone, the production of ROS is decreased during the oxidation of complex I substrate, in parallel with the preservation of mitochondrial content and the rate of oxidation through cytochrome c oxidase (55
). This mitochondrial “protective” response to complex I inhibition during ischemia has been replicated by the use of amobarbital (a reversible inhibitor of complex I) (43
), by S-nitrosoglutathione–dependent transnitrosation in the Langendorff perfused heart (39
), and in this study, by the naturally occurring anion nitrite. Collectively, these data suggest that transient inhibition of complex I in the I/R milieu can have protective effects associated with reduced ROS production during reperfusion. In contrast, chronic inhibition of complex I under an otherwise physiologic milieu can be cytotoxic and associated with elevated ROS production (56
Interestingly, nitrite appears to be unique among the aforementioned agents in that it does not inhibit complex I activity during normal physiology but only inhibits complex I activity in the setting of I/R injury. These data suggest that the modification of complex I by these near physiological concentrations of nitrite is dynamic and only operational in the setting of ischemia. The mechanisms whereby low-dose nitrite-mediated modification of complex I is “fine-tuned” to have no appreciable inhibitory effect under normal homeostatic conditions but sufficient inhibitory effects in the context of hypoxic or redox stress are currently unknown and require further investigations. However, such dynamic inhibition of complex I allows for sustained mitochondrial oxidative phosphorylation through complex II during I/R while limiting ROS generation, mitochondrial calcium overload, and the release of cytochrome c (37
The chemistry of nitrite-dependent nitrosation of complex I will also require further study, considering the fact that the NO radical will not directly nitrosate reduced thiol (one-electron oxidation is required). There are several possibilities that might be unique to the mitochondria. First, direct nitrosation of thiols by nitrous acid: the inner membrane space has an estimated pH of ~7.2, with the matrix having a pH of ~7.9. Although minimal nitrous acid should form at pH 7.2 (the pKa of nitrite is ~3.2), any nitrous acid that formed would occur in the inner membrane space and potentially at complexes I, III, IV, and V, which actively pump protons. The hydrophobic transmembrane environment of these complexes would stabilize both nitrous acid, N2
, and the formed SNO. Second, NO production from nitrite reduction could secondarily react with nitrogen dioxide or superoxide formed from mitochondrial oxidation pathways and could form nitrosating intermediates such as N2
. Third, we have recently found that nitrite binds to and reacts with metheme proteins, such as methemoglobin, to produce an NO2
radical–like intermediate that can then react with NO to form N2
would directly nitrosate complex I. This would provide a metal-based pathway to S-nitrosation by nitrite. Although these pathways remain theoretical, the mechanisms of S-nitrosation by nitrite are currently an active area of research in the NO field.
It is tempting to speculate that nitrite is the endogenous molecule that regulates ischemic ROS formation at complex I after reperfusion. Such an innate mechanism could represent an evolved response to limit tissue injury during birth (for the fetus and mother), traumatic hemorrhage, and extreme exercise. From a therapeutic standpoint, nitrite would represent an ideal candidate to confer mitochondrial and, by extension, cellular resilience to I/R injury through the partial inhibition of complex I. The therapeutic potential of nitrite is underscored as follows: (a) this anion is a chemically stable endogenous reservoir for NO, (b) the required therapeutic concentration is in the nanomolar range, and (c) it is also readily available in diet (i.e., the Mediterranean diet) and, as shown in our study, its protective effect is clearly observed with oral intake.
Consistent with an innate physiological role for nitrite in modulating the stress response, the cytoprotective effects of nitrite in the heart and liver are measurable at nitrite doses <1.2 nmol in murine models of myocardial infarction and hepatic I/R (8
). These doses increase plasma nitrite levels by <10% and are consistent with increases observed after the ingestion of a standard leafy green salad (47
) or after regular moderate exercise (59
). Furthermore, mice with diminished basal plasma nitrite concentrations are more susceptible to I/R injury, an effect that is attenuated by administration of exogenous nitrite (11
). These data suggest that a diet rich in nitrate and nitrite may have profound cytoprotective effects and, most provocatively, could constitute the “active” ingredient of the cardioprotective Mediterranean diet (47
In conclusion, we have shown that nitrite potently mediates cytoprotection after I/R of the mammalian heart and liver at the mitochondrial level through the transient inhibition of complex I and subsequent limitation of oxidative damage. The acute and remote temporal windows of nitrite-dependent cytoprotection suggest that nitrite may be a central mediator in ischemic preconditioning. Collectively, these studies reveal a global role for nitrite in the regulation of ischemic responses at the subcellular level. Finally, its efficacy as an oral preparation at near physiologic doses suggests a role for nitrite as a dietary cardioprotective agent.