The present data indicate that nitrite protects the heart against injury from infarction and decreased post-ischemic ventricular function in both in vivo
and in vitro
models of ischemia/reperfusion. This effect of nitrite is dose- and concentration-dependent with optimal protection occurring at 4 mg/kg I.V. in vivo
and 10 μM in vitro.
Nitrite conferred cardioprotection over the range of 10–100 μM in vitro.
One hundred micromolar nitrite also vasodilates aortic ring preparations [21
], with subsequent studies showing the effect is mediated by soluble guanylyl cyclase [22
]. These observations suggested nitrite is being reduced to nitric oxide which elicits protection. Our studies on cardioprotection by nitrite are in strong agreement with the studies of Webb et al [7
] and Duranski et al [6
] and in addition describe novel findings on the mechanisms underlying nitrite-induced cardioprotection.
We calculated the nitrite concentration in the plasma at the time the heart becomes ischemic 15 minutes after nitrite administration. Previous pharmacokinetic studies of nitrite in the rat [24
] predict a plasma concentration of 30 μM 15 minutes after intravenous administration at the optimal dose of 4 mg/kg. Thirty micromolar nitrite conferred cardioprotection in our in vitro
studies. Thus the optimal dose of 4 mg/kg for the in vivo
studies is within the range of concentrations (10–100 μM) for nitrite that confer protection in the in vitro
Nitric oxide is an important defense against injury from myocardial ischemia/reperfusion. Generation of nitric oxide from L-arginine by nitric oxide synthase requires molecular oxygen as an essential substrate. Under conditions of oxygen deprivation, as encountered during ischemia, oxygen levels are rapidly depleted so that nitric oxide synthase activity will be inhibited. Generation of nitric oxide from the reduction of nitrite under ischemic conditions may represent an important mechanism to maintain nitric oxide production for cell survival and function until aerobic conditions are restored, following reperfusion, so that nitric oxide synthase can resume production of nitric oxide. In control studies, nitrate administered in an identical protocol did not confer cardioprotection in both in vivo and in vitro models of myocardial ischemia/reperfusion indicating that the protective effect is specific to nitrite.
Xanthine oxidoreductase is a molybdoflavin enzyme present in the highest concentrations in gut, liver and breast milk as well as in plasma from patients with inflammation [25
]. Xanthine dehydrogenase is responsible for the conversion of xanthine to uric acid. Xanthine dehydrogenase activity can be converted to xanthine oxidase, under reducing conditions present during ischemia, and it is the oxidase form of the enzyme that is responsible for superoxide production. The activity of xanthine dehydrogenase in the rat hearts we studied accounts for approximately 80% of total enzymatic activity with the balance contributed by xanthine oxidase. This distribution in catalytic activity between the two forms of the enzyme is in agreement with previous studies on xanthine oxidoreductase activity [26
]. The protective effects of nitrite against infarction and decreased post-ischemic ventricular dysfunction were abolished by oxypurinol, an inhibitor of the molybdo-pterin sites of xanthine oxidoreductase. This site is responsible for the binding and oxidation of purine substrates and has also been shown, in in vitro
studies to be the site of nitrite reduction [28
]. Surprisingly, the effect was also inhibited by DPI, an inhibitor of flavoprotein reductases, which will block electron flow from the molybdenum site to electron acceptors (oxygen or NAD+
). It has been shown previously that DPI is unable to inhibit nitrite reduction by xanthine oxidoreductase using xanthine as a substrate, but could inhibit NADH-driven nitrite reduction [28
]. However, DPI is a relatively non-specific inhibitor and it is possible that other flavoprotein reductase enzymes are contributing to nitric oxide formation. We show that apocynin, an inhibitor of the NADPH oxidase, a flavoprotein reductase [29
], abolishes nitrite-induced cardioprotection. This novel finding may explain why DPI abolishes the protective effect of nitrite and identifies a component of another mechanism by which nitrite may confer its salubrious actions. Although the reduction of nitrite by NADPH oxidase has not previously been observed, the reduction of nitrite by ferrous heme proteins, such as hemoglobin, is well established and may be mechanistically similar [30
Our study suggests that nitrite can act as a substrate for both xanthine dehydrogenase and xanthine oxidase during ischemia to produce nitric oxide. Harrison has also shown that nitric oxide formation results from reduction of nitrites, both inorganic [16
] and organic [32
], catalyzed by xanthine oxidoreductase. Activity of xanthine oxidase and xanthine dehydrogenase is increased by nitrite during ischemic conditions, where oxygen and pH are reduced, but not during aerobic conditions. Increased xanthine oxidoreductase activity as a consequence of ischemia has been reported previously [33
]. Alternately, ischemia may repair the inactive desulfo form of the enzyme to the fully active form as previously suggested [25
The spin trap carboxy-PTIO abolished nitrite-induced cardioprotection indicating that nitric oxide is responsible for protection against post-ischemic ventricular dysfunction and necrosis. Inhibition of the enzyme nitric oxide synthase with L-NMA did not abolish cardioprotection by nitrite, suggesting nitrite-induced cardioprotection occurs by a nitric oxide synthase-independent mechanism. In addition it has been shown that NOS itself may function as a nitrite reductase in the absence of oxygen [37
], but the lack of effect of L-NMA rules out this possibility in the isolated cardiac preparation used in these studies.
The signaling pathway by which nitrite protects against injury from ischemia/reperfusion has not been established. We show here that nitrite-mediated protection is mediated in part by the activation of KATP
channels. Currently there are two KATP
channels thought to be present in the cardiac myocyte, one in the sarcolemma (sarc KATP
channel) and one in the inner mitochondrial membrane (mito KATP
channel). Our data show that the sarcolemmal KATP
channel appears to act as trigger and the mitochondrial KATP
channel as an effector of nitrite-induced cardioprotection. There is evidence to suggest that opening either channel may be important in producing cardioprotection, although the bulk of evidence suggests that it is the mito KATP
channel that is responsible for mediating the protective effect of ischemic preconditioning [39
]. The role of additional components in the signaling pathway by which the heart is protected by nitrite against injury remains unknown.
Furthermore, we found that a single treatment with nitrite before ischemia reduces cardiac necrosis and increases the recovery of ventricular function following reperfusion. The cardioprotective effect of nitrite is observed immediately after treatment suggesting that the production of new protein is not necessary to manifest its cardioprotective effect. Nitrite also confers protection against injury when given after the onset of ischemia, i.e. after the onset of symptoms. In patients experiencing symptoms of a myocardial infarction, or those who are about to undergo cardiac surgery, nitrite could be administered acutely to decrease injury to the heart from ischemia/reperfusion. Thus, nitrite may represent an important and potent agent for immediately conferring cardioprotection. Nitrite is well tolerated and does not require an elaborate drug delivery system as is needed for several gene-based therapies to confer cardioprotection.
Sodium nitrite, a naturally occurring chemical and common meat preservative, is only used medically to treat cyanide poisoning. Our study suggests this chemical can be used to protect and preserve organs such as the heart in the setting of myocardial infarction and preservation of the heart for transplantation. In support of this notion, nitrite is undergoing clinical trials to increase nitric oxide production in patients with coronary artery disease [41
] and in preconditioning mediated tolerance to ischemia [42
]. These clinical trials will determine whether dietary nitrite and nitrates can produce nitric oxide to dilate peripheral blood vessels and whether protection by nitrite is equivalent to preconditioning in the setting of ischemia/reperfusion in the human forearm.
We conclude that nitrite, a simple inorganic anion, is reduced by xanthine oxidoreductase to nitric oxide to increase resistance of the heart to injury from ischemia/reperfusion. The cardioprotective effects of nitrite are also mediated by NADPH oxidase and KATP channels suggesting nitrite acts by triggering multiple pathways. These observations contribute to a re-evaluation of how we view the mechanisms and biological effects of nitrite.