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. 2007 Sep 3;204(9):2089-102.
doi: 10.1084/jem.20070198. Epub 2007 Aug 6.

Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer

Sruti Shiva  1 , Michael N Sack, James J Greer, Mark Duranski, Lorna A Ringwood, Lindsay Burwell, Xunde Wang, Peter H MacArthur, Amir Shoja, Nalini Raghavachari, John W Calvert, Paul S Brookes, David J Lefer, Mark T Gladwin
Affiliations

Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer

Sruti Shiva et al. J Exp Med. .

Abstract

Nitrite (NO(2)(-)) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II-IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.

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Figures

Figure 1.
Figure 1.
Nitrite mediates both acute and delayed cytoprotection after I/R injury to the heart and liver. (A) Model of hepatic ischemia and myocardial infarction in which nitrite or saline was administered either 24 h before or during ischemia. (B) Plasma ALT levels 5 h into reperfusion in mice after sham surgery, sham surgery with nitrite treatment, ischemia with acute nitrite treatment, or ischemia with nitrite preconditioning (PC). (C) Infarct size as a percentage of area at risk 24 h after myocardial infarction in the absence of nitrite (I/R), with nitrite treatment 5 min before reperfusion (acute), or nitrite treatment 24 h before myocardial infarction (PC). (D) Representative sections of myocardium stained with Evan's blue and triphenyltetrazolium chloride 24 h after infarction in mice receiving no nitrite (I/R + vehicle), acute nitrite treatment, or nitrite preconditioning. *, P < 0.05 in comparison with the I/R + vehicle group; **, P < 0.01 versus the I/R + vehicle group.
Figure 2.
Figure 2.
Nitrite preconditioning protects from ischemic damage at the mitochondrial level. (A) Respiratory rates (in the presence of succinate and ADP) of liver mitochondria isolated from mice 5 h after being subjected to hepatic I/R in the presence and absence of acute and preconditioning nitrite. (B) In vitro model of mitochondrial anoxic/reoxygenation. Red trace is mitochondria subjected to anoxia. Blue trace is mitochondria subjected to normoxia for 30 min. (C) Representative respiration traces of 1 mg/ml of mitochondria isolated from rats preconditioned with saline (continuous lines) or 480 nmol nitrite (dashed lines) before (green) and after (blue) anoxia. (D and E) Postanoxic respiration (D) and ATP synthesis (E) rates expressed as a percentage of preanoxic rate for mitochondria isolated from rats preconditioned with saline or nitrite. All experiments are means ± SEM of at least n = 3 independent experiments. *, P < 0.01.
Figure 3.
Figure 3.
Nitrite-dependent cytoprotection does not modulate mitochondrial biogenesis. Rats were given one intraperitoneal injection (480 nmol) of saline or nitrite daily for nine consecutive days. (A and B) Recovery of respiration (A) and ATP (B) generation rates of mitochondria isolated from these rats after 30 min of anoxia in vitro (white, saline; green, nitrite). (C) Relative expression of genes in the livers of nitrite-treated rats on day 9 presented as the fold change in gene expression compared with saline-treated rats. (D) Protein expression of the 39-kD subunit of complex I, cytochrome c, and ATPase subunit B in the livers of rats after 9 d of nitrite or saline treatment. All experiments are means ± SEM of at least n = 3 independent experiments. *, P < 0.01.
Figure 4.
Figure 4.
Acute nitrite treatment protects mitochondria against I/R injury. (A) Model of in vitro mitochondrial damage showing preanoxic (green) and postanoxic rates in the absence (blue) and presence (red) of nitrite. Arrow denotes the addition of 10 μM nitrite. Respiration rates (B) and ATP generation rates (C) before anoxia (green) and after anoxia in the presence of saline (blue) and nitrite (red). (D) Quantification of rate of ATP generation before and after anoxia. (E) Recovery of respiration rate with increasing concentrations (0–100 μM) of nitrite added during anoxia. All experiments are means ± SEM of at least n = 3 independent experiments. *, P < 0.01. RLU, relative light units.
Figure 5.
Figure 5.
Nitrite treatment inhibits complex I. (A) Recovery of complex II–dependent respiratory rate in mitochondria that were untreated (Ctrl), treated with 10 μM nitrite alone, 200 μM diazoxide (Diaz), diazoxide and nitrite, 150 μM glibenclamide (Glib), or glibenclamide and nitrite during anoxia. (B) Recovery of respiration of mitochondria respiring on succinate (complex II) or glutamate/malate (complex I) and ADP in the absence (green) and presence (red) of 10 μM nitrite during anoxia/reoxygenation. (C) Absolute respiratory rates before and after anoxia of mitochondria from rats treated with nitrite (green) or saline (red) 24 h earlier. (D) Respiratory rates of mitochondria isolated from mice 5 h after they were subjected to hepatic I/R in vivo. (E) Complex I activity of isolated mitochondria treated with increasing concentrations of nitrite. (F) Recovery of complex I respiration rate in untreated mitochondria or treatment with 20 μM nitrite, 100 μM PTIO, or nitrite and PTIO. All experiments are means ± SEM of n = 3 experiments. *, P < 0.01.
Figure 6.
Figure 6.
Nitrite S-nitrosates complex I. (A) Representative chemiluminescence trace of nitrite-treated mitochondria injected into triiodine before and after pretreatment with mercuric chloride (+Hg). (B) Quantitation of S-nitrosation in mitochondria from A (red) and an identical experiment using copper (I)/cysteine as a reductant instead of triiodine (blue). (C) Representative copper (I)/cysteine–based chemiluminescence trace of mitochondria isolated from rats treated with 480 nmol of saline or nitrite 24 h earlier. (D) Quantification of traces similar to those shown in C. (E) Protein was extracted from rat liver mitochondria and loaded onto a Superose 6 size-exclusion column. The SNO and complex I activity of each fraction was then determined and are represented as picomoles of SNO per fraction and complex I activity of the neat fractions, respectively. Mitochondria subjected to ischemia without NO2 − treatment resulted in no SNO detection (not depicted). All experiments are means ± SEM of at least n = 3 independent experiments. *, P < 0.01.
Figure 7.
Figure 7.
Nitrite decreases oxidative damage in mitochondria. (A) Representative traces of ROS production measured by amplex red in mitochondria without substrate (light blue) in the presence of glutamate/malate and ADP before anoxia/reoxygenation (green), and after anoxia/reoxygenation in the presence (red) and absence (blue) of 20 μM nitrite. (B) Aconitase activity of mitochondria during normoxia and after anoxia/reoxygenation in the presence and absence of 20 μM nitrite. (C) Aconitase activity in liver tissue from mice 5 h after they were subjected to hepatic I/R or sham surgery in the presence and absence of 48 nmol of acute or preconditioning nitrite. (D) Representative traces of calcium-induced pore opening of mitochondria subjected to anoxia/reoxygenation in the presence of 20 μM nitrite. Traces are in the absence of calcium (black), in the absence of nitrite treatment (red), or with nitrite treatment (blue). (E) Western blot analysis of cytochrome c remaining in mitochondria from the conditions shown in D. All experiments are means ± SEM of at least n = 3 independent experiments. *, P < 0.01; #, P < 0.05.
Figure 8.
Figure 8.
Hepatic and myocardial I/R injury are attenuated in mice after oral nitrite therapy. (A) The effects of oral nitrite preconditioning (PC) on the severity of hepatic I/R injury (measured as aspartate aminotransferase [AST] and ALT) in mice. Mice received nitrite via oral gavage at 24 h before 45 min of hepatic ischemia and 5 h of reperfusion. Numbers for each group are shown inside the bars. (B) Bar graph of oral nitrite preconditioning and myocardial I/R injury in mice. Mice received oral nitrite at 24 h before 30 min of ischemia and 24 h of reperfusion. The myocardial area-at-risk (AAR) per total left ventricle (LV) was not significantly different between study groups (NS). The myocardial infarct size (Inf) per area-at-risk was significantly (P < 0.0001) reduced in the oral nitrite preconditioning group when compared with the oral vehicle group. Eight animals were investigated in each group.
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