This study demonstrates that the anion nitrite (NO2−) potently limits myocardial infarction and apoptosis in the reperfusion phase of injury. The mechanism of myocardial protection is independent of the time-ischemia severity integral since a brief 5-minute infusion of nitrite during the end of a two-hour occlusion reduced infarct size and apoptosis almost as much as a 60-minute infusion and the short infusion caused virtually no hemodynamic perturbations. The improved myocardial salvage associated with the 5-minute nitrite infusion was not explainable on simple hemodynamic factors such as preload, afterload, rate pressure product, or the area at risk. Both nitrite infusion protocols had beneficial effects on global left ventricular function and minimized endocardial “no-reflow” phenomenon, characterized by microvascular obstruction in the infarct core. Therefore, we conclude that nitrite provides a direct cellular cardioprotective mechanism in the reperfusion phase of injury. Furthermore, nitrite can provide this remarkable degree of cardioprotection on a time scale compatible with intravenous adjunctive therapy to acute percutaneous interventions for acute myocardial infarction.
Two recent studies suggest that nitrite potently limits ischemia-reperfusion cytotoxicity with a maximal effect observed at low concentrations 14,15
. While the protective effect was maximal at blood concentrations of 10 micromol/L (48 nmole dose for a mouse), even doses as low as 1.2 nmoles - which were associated with increases in blood levels of nitrite from 700 nmol/L to only 900 nmol/L, reduced the infarction size by 50% 15
. The cytoprotective effect of nitrite reduced apoptosis and was associated with intracellular reduction of nitrite to NO, independent of the NO synthase and hemeoxygenase 1 enzymes. In the current study, this cytoprotective effect is recapitulated in a large mammal exposed to a longer ischemic time and more extensive infarction relative to area at risk. Remarkably, a five-minute infusion of nitrite in the current study increased plasma levels of nitrite in dogs from a ~1 umol/kg at baseline up to 5 umol/L, with no associated increases in plasma or red cell S-nitrosothiols. These near-physiological increases in nitrite decreased myocardial infarction size from 70 to 20% of the area at risk, and improved cardiac contractile function.
Nitric oxide that diffuses into blood rapidly reacts with both oxy- and deoxyhemoglobin to form methemoglobin/nitrate and iron-nitrosyl-hemoglobin (HbFeII
-NO), respectively 25,36
. These reactions shorten half-life of NO in blood to less than 2 milliseconds and thus maintain endothelial-derived NO as a paracrine vasoregulator 37,38
While NO per se is inactivated by reactions with hemoglobin, it may be stabilized in blood by the formation of NO modified proteins, peptides and lipids, and oxidation to the anion nitrite. It is increasingly clear that a number of intravascular chemical NO-modified species are capable of mediating vasodilation, including S-nitrosothiols 16,17
, nitrite 1,3,18,19
, N-nitrosamines 21-24
, iron-nitrosyls 25
, and recently identified nitrated lipids 26-29
Both human blood flow experiments and studies of ischemia-reperfusion over the last two years suggest that nitrite is one of the major endocrine NO species in blood. In earlier physiological studies, we observed artery-to-vein gradients in nitrite across the human forearm, with increased consumption of nitrite during exercise stress, suggesting that nitrite is metabolized across the peripheral circulation 1
. While nitrite was considered biologically inert, we found that nitrite induced concentration-dependent vasodilation healthy human volunteers 3
. Nitrite levels even as low as 900 nmol/L produced vasodilation in humans during exercise stress with concurrent NO synthase inhibition with L-NMMA suggesting a physiological role for nitrite in vascular homeostasis 3
. The potent vasodilating effects of nitrite have been verified in a number of models 12,14,19,20,39
The degree to which nitrite-induced vasodilation and coronary collaterals contribute to myocardial protection warrants consideration. It would require a very large sample size to determine the extent to which the statistically insignificant increase in microsphere blood flow (60-minute nitritre group) contributes to myocardial protection since the magnitude of effect is very small. In the 5-minute nitrite group, nitrite-induced vasodilation cannot significantly alter the net deficit in the time-blood flow integral and thus is biologically unlikely to confer protection by a mechanism related to reduced ischemia as a result of collateral blood flow. However, collateral blood flow may provide a route for nitrite to reach into the ischemic myocardium and thus indirectly facilitate protection afforded by mechanisms that directly modulate the biochemical mechanisms underlying ischemia reperfusion injury.
The cardioprotective effects of nitrite infusion in the current study were associated with specific increases in plasma nitrite at near physiological concentrations. Although the 60-minute infusion of nitrite was associated with increases in both plasma nitrite and blood S-nitrosothiols, only nitrite levels increased during the five-minute infusion protocol. These data support the thesis that nitrite is an endocrine intravascular NO-species that modulates systemic response to hypoxic/ischemic injury.
During cardiac ischemia and reperfusion, nitrite in tissue is reduced to NO and forms iron-nitrosylated (Fe+2-NO) and S-nitrosated modified proteins, via reactions with deoxymyoglobin and other cellular heme proteins3,10,40,41
. The rapid, facile metabolism of nitrite to NO with subsequent modification of target proteins has been documented in the heart and liver during both regional and global IR injury15,42,43
. The formation of NO in the heart during ischemia has been documented using electron paramagnetic resonance and liquid and gas phase chemiluminescence. We have recently found that nitrite will specifically post-translationally S-nitrosate complex I of the mitochondrial electron transport chain (METC); this effectively reduces electron flow through the METC and reduces reactive oxygen species formation during reperfusion44
. This damping or tuning of electron transport inhibits opening of the mitochondrial permeability transition pore, decreases cytochrome C release, and limits apoptosis. The nitrite-dependent decrease in TUNNEL staining is consistent with this mechanism of cytoprotection. Other intracellular targets for S-nitrosation by nitrite during IR exposure could include the L-type calcium receptor45
. In addition, stabilization of myglobin as iron-nitrosylated myoglobin may limit heme based oxidation reactions in the cardiomyocyte15
In this study, we have shown an increase in iron-nitrosylation with nitrite treatment. While this increase reflects nitrosylation of heme proteins, such as myoglobin, it may also indicate nitrosylation of non-heme iron. Cellular non-heme iron content plays a role in determining the sensitivity of cells to NO-mediated apoptosis46
, with increasing concentrations of non-heme iron rendering cells less susceptible to NO-mediated apoptosis. Non-heme iron is able to bind NO (forming Fe-NO) which decreases the bioavailability of NO, as well as oxidizes NO to NO+ to promote S-nitrosothiol formation (including the S-nitrosation of caspases). In the case of nitrite, if nitrite is reduced to NO, which then mediates S-nitrosation of tissue components to illicit cytoprotection, tissue non-heme Fe would catalyze S-nitrosothiol formation and promote the anti-apoptotic effects of nitrite. This may be consistent with the increase in Fe-NO seen in tissues after nitrite administration during ischemia-reperfusion.
While current reperfusion therapies are efficacious in the treatment of acute MI, intrinsic and practical delays between symptom presentation and intervention compromise the amount of myocardial salvage. Despite great advances in percutaneous coronary interventions that result in excellent restoration of coronary blood flow, the mortality after MI remains at 7% and virtually all patients suffer some degree of myocardial necrosis. The extent of the myocardial infarction predicts future cardiac function. Post MI heart failure represents a huge burden on our health care system. Adjunctive pharmacological therapies that improve the amount of myocardial salvage following reperfusion of an acute MI could positively impact cardiac function and possibly prognosis. Such adjunctive therapies should possess the following characteristics: a) significant cardioprotection after prolonged ischemia; b) simple administration; c) low expense; d) low dose required for pharmacological action; e) short half-life and rapid onset of action; f) minimum associated regional and systemic side effects; and g) a cardioprotective mechanism that is not dependent on vasodilation or changing rate pressure product. Nitrite satisfies these requirements.
There are limits to the current study. While it would have been desirable to also study cardioprotection with 2 doses of nitroglycerin, this was not practical for sample size considerations due to the large number of potential comparisons. Nitrite did provide better cardioprotection than nitroglycerin in a mouse model15
and inhaled NO was more potent than nitroglycerin in a swine model47
. Even in the current set of experiments, there is limited power to detect differences between groups. Thus, one must interpret statistics showing no change between groups with caution. However, the key findings that nitrite provides cardioprotection and reduces infarct size are supported with statistical confidence. Furthermore, biological factors that modulate infarct size such as rate pressure product, residual perfusion during ischemia, and systolic wall stress all indicate that the 5-minute nitrite group faced as challenges that directionally should have lead to larger infarcts than the control group.
In conclusion, nitrite (NO2−) possesses the characteristics of an ideal adjunctive therapy for acute MI. From a feasibility perspective, nitrite can be administered intravenously and the 5-minute dose does not significantly alter hemodynamics. In patients with acute MI, the 5-minute infusion of nitrite could be initiated on arrival to the catheterization laboratory shortly prior to percutaneous coronary intervention.