Is nitrite the circulating endocrine effector of remote ischemic preconditioning?

Nitric oxide signaling

Nitric oxide (NO) is a highly diffusible, free radical signaling molecule that is produced by the endothelial NO synthase enzyme, which converts L-arginine and molecular oxygen into L-citrulline and NO [1, 2]. Nitric oxide diffuses from the endothelium to the smooth muscle where it binds with high affinity to the heme group of soluble guanylate cyclase, which in turn catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) [3]. Nitric oxide signaling is largely paracrine, with potential endocrine effects limited by its radical nature and extremely high reactivity with other heme containing proteins such as hemoglobin and myoglobin [4]. When NO encounters oxyhemoglobin in blood or oxymyoglobin in cardiomyocites it reacts at rates near the diffusion limit to form nitrate and methemoglobin (dioxygenation reaction) [5,6]. It will also react with the deoxyhemes of these proteins to form iron-nitrosyl-complexes, which can release NO but quite inefficiently via the oxidative denitrosylation reaction [7]. These two reactions, dioxygenation and iron-nitrosylation, prevent NO from forming in the endothelium and diffusing to distant organ targets, such as the heart, intestine, kidney, brain or liver.

Despite the strict paracrine limitations imposed by this chemistry, a number of studies suggested that endocrine NO signaling is possible. The Kubes group showed that NO delivered by inhalation to cats could improve blood flow and limit inflammation in the cat intestine subjected to ischemia-reperfusion (I/R) injury [8]; Gladwin and Cannon later showed that this was possible in the human circulation [6]. Many subsequent studies have shown that inhaled NO could rescue distal organs from I/R injury and infarction. In fact, upregulation of eNOS selectively in the heart could rescue the liver from I/R injury [9]. However, free NO cannot account for these effects based on the short half life of NO in blood, on the order of 2 milliseconds or less [10].

Many investigators have examined reaction products of NO in blood, attempting to devine the mediator of endocrine NO signaling. While S-nitroso-albumin and S-nitrosohemoglobin were first proposed as endocrine NO metabolites, the levels of these species even during NO inhalation are quite low, using validated chemiluminescent detection methods [4]. Human studies with NO inhalation suggested that the NO oxidation product nitrite (NO₂^-) increases significantly, with arterial levels higher than venous levels, suggesting this anion could account for the effect [4, 6, 11]. Unlike authentic NO, nitrite has a half life in mammals approaching 60-minutes [12]. Infusions of nitrite in humans and animal models indicated that nitrite was a potent vasodilator and cytoprotective agent that could mimic all the observed effects of NO inhalation [13-16]. Recent studies have carefully repleted nitrite levels to those observed with NO inhalation and produced similar reductions in organ infarction volumes, confirming the role of nitrite as the endocrine effector of inhaled NO [17].

Elusive endocrine mediator of remote ischemic preconditioning

Another line of investigation suggests the existence of an endocrine mediator of organ cytoprotection during remote ischemic preconditioning (rIPC). The idea that a signal transduction exists between the local site of remote ischemia and the myocardium was demonstrated by Przyklenk et al. in the early 1990’s. They found, using a canine model, that brief episodes of ischemia and reperfusion in the circumflex coronary artery reduce the size of the myocardial infarct arising from the occlusion of the left anterior descending artery [18]. This form of myocardial protection was subsequently found to occur with “remote” ischemia and reperfusion of non-cardiac organs. Transient ischemia of a variety of tissues such as kidney, small bowel, liver, skeletal muscle and even brain induces a systemic protective effect against the subsequent extended I/R injury of the myocardium [19-21]. Such phenomenon was termed “preconditioning at a distance” [22] and appears to be highly conserved across species. Animal studies with transplanted hearts further support the role of a circulating substance or a group of transduction mediators with protective effects against I/R injury. Remote limb preconditioning of a pig that received a donor heart was able to reduce myocardial infarct size [23] and hearts excised from a rat that had been subjected to remote limb preconditioning experienced a smaller infarct size when subjected to sustained I/R on a Langendorff-apparatus [24].

The finding that a reperfusion period of the remote preconditioned organ is required after the brief ischemia suggests that the reperfusion period may be needed to “washout” a humoral factor generated by the preconditioning ischemia, which is then transported to the heart [21]. Many experimental studies have attempted to identify the nature of the endocrine mediators circulating in the blood stream which conveys the preconditioning signal from the remote organ to the heart [25-27]. However the actual identity of the humoral mediator remains unknown.

NO and cardioprotection

There is a large body of literature describing the protective properties of NO as an element of the cytoprotective factor, despite the limitations of endocrine movement to a remote site. Endogenous NOS-derived NO appears to play a pivotal role in mediating the protective effect of hindlimb rIPC in reducing liver damage and this is abrogated by treatments with the NO scavenger cPTIO and inhibited in the endothelial NO synthase knockout mouse [28]. Tokuno et al. [20] have implicated iNOS activation as a trigger for delayed rIPC of the heart using cerebral ischemia as preconditioning stimulus. The cardioprotective effect was seen 24 hours later and was absent in iNOS knockout mice. Further studies demonstrated that NO is necessary for the development of ischemia-induced delayed protection against both myocardial stunning and myocardial infarction [29]. While it is clear that NO synthase and NO appears to participate in the process of rIPC, the mechanism for NO transport to a distant site in this process, and the very nature of the “endocrine” rIPC mediator has remained a mystery.

Nitrite as endocrine mediator of rIPC

In the current issue of Circulation Research, Rassaf and colleagues investigate the mechanism of remote ischemic preconditioning (rIPC) and explore the possible identity of the circulating “endocrine” mediator [30]. They first find in human studies that, similar to the case with inhaled NO exposure, the levels of plasma nitrite increase after shear mediated eNOS activation during brachial artery occlusion and release (reactive hyperemia). This is caused by eNOS activation with NO formation and oxidation to the more stable nitrite. They confirm this by blocking the high flow shear associated with reactive hyperemia by using partial 50% compression of the brachial artery following ischemia. This is a very creative control, allowing for regional ischemia without the shear-induced activation of eNOS and formation of intravascular nitrite.

They then do remote ischemia preconditioning (rIPC) studies in the legs of mice and show that nitrite levels increase. Inhibition of NO with PTIO or in eNOS KO mice, prevents the rise in nitrite and rIPC effects on myocardial infarction. This association is mechanistically confirmed by infusions of nitrite to match elevated levels observed with rIPC. Finally, they infuse human plasma with and without rIPC into the isolated heart model of IR and show that elevations in nitrite (removed with acidified sulfanilamide and repleted) account for effects. Overall, the studies are highly translational and utilize creative methodologies to test a major pathway in biology, the process and effector of remote ischemic preconditioning.

Myoglobin as nitrite reductase

While these studies suggest that nitrite forms during rIPC and travels in the plasma to the heart, how is it then converted in the heart back into cytoprotective NO? During ischemia nitrite is reduced to NO and N₂O₃ by different nitrite reductase enzyme systems [31, 32].Mitochondrial NO and S-nitrosothiols formed from nitrite dynamically and reversibly inhibit complex I during reperfusion, which limits ROS formation from complex I and III [33, 34]. This ultimately prevents the opening of the mitochondrial permeability transition pore and the release of cytochrome c. It has recently been shown that the site of nitrosation is on Cys 39 of the ND3 subunit of complex I [34]. A number of enzymes are required to convert nitrite into NO during organ ischemia. For example, in the heart, deoxygenated myoglobin acts as a functional nitrite reductase [35] (Figure 1). Nitrite-dependent NO formation is significantly decreased in myoglobin deficient hearts [36] and nitrite administration reduces myocardial infarction with abrogated effects in the myoglobin knockout mice [37]. In the current study Rassaf and colleagues show that the effect of rIPC is inhibited in the myoglobin knock-out mouse, providing further support that the endocrine mediator of this effect is nitrite, which is produced in the extremity, travels in blood to the heart, where it is reduced by myoglobin to produce NO.

Figure 1.

Mechanisms of nitrite-mediated cytoprotection. In the cardiomyocytes nitrite is reduced to NO by reactions with deoxy-myoglobin and then can react with and inhibit complex I of the mitochondrial electron transport chain. This inhibition is reversible and occurs immediately during reperfusion to limit reactive oxygen species formation and to prevent the release of cytochrome c. Figure adapted from Bueno et al, “Nitrite signaling in pulmonary hypertension: mechanisms of bioactivation, signaling, and therapeutics.” Antioxidants & redox signaling, 2013. 18(14): p. 1797-809.

Conclusion

A potential limitation of the current study is the reliance on mouse models of myocardial infarction to test the cytoprotective effects of nitrite. A recent clinical trial was presented at the 2013 American Heart Association meetings investigating the therapeutic effects of nitrite in ST-elevation myocardial infarction (STEMI) showed that sodium nitrite administered prior reperfusion does not reduce infarct size [38]. Evaluation of the full results of this trial will be required to understand the dose, timing, plasma nitrite levels achieved and fidelity of the study design. However, these results are likely to raise questions as to the relevance of findings from mouse models of ischemia-reperfusion injury to human disease.

In summary, this study provides compelling evidence that limb ischemia causes metabolic vasodilation that leads to increased blood flow and shear-force on the endothelium of conductance blood vessels to activates eNOS. Activated eNOS produces NO which is oxidized in plasma to nitrite. Nitrite then circulates as the endocrine mediator of rIPC and travels to the heart. Finally, when the heart is subjected to ischemia the nitrite is then reduced by deoxymyoglobin to form NO in the cardiomyocyte, limiting cellular injury and infarction.