Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury

Abstract

The nitrite anion is reduced to nitric oxide (NO*) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO* signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type ((+/+)) and knockout ((-/-)) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO* generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO* generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO* by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin(+/+) but not in myoglobin(-/-) hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin(+/+) mice, whereas in myoglobin(-/-) mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation [corrected]

Fig. 1.

NO^• production from the ex vivo retrograde perfused heart during ischemia by using the NO^• collection heart chamber. (A) Representative time courses of NO^• production from nitrite during 30 min ischemia in myoglobin wild-type and knockout hearts measured by chemiluminescence every 15 min. (B) NO^• generation in myoglobin^+/+ and myoglobin^−/− hearts after 15 and 30 min of ischemia. Application of nitrite (100 μM) showed a time-dependent increase of NO^• production in myoglobin^+/+ hearts from 15 ± 3 ppb to 25 ± 1 ppb (P < 0.05; n = 3), whereas in myoglobin^−/− hearts, a decrease from 16 ± 2 ppb to 12 ± 2 ppb was observed (P < 0.05; n = 3). (C) Representative traces of NO^• generation by heart homogenates from myoglobin^+/+ and myoglobin^−/− mice treated with nitrite (1 mM). (D) Quantitation of nitrite generation from several curves similar to C (P < 0.05; n = 4). *, P < 0.05.

Fig. 2.

Nitrite-induced formation of NO-heme complexes during ischemia and reperfusion. (A) Experimental protocol. (B Upper) EPR spectrum of the mitochondrial ubiquinone radical signal formed in a postischemic reperfused myoglobin wild-type (^+/+) heart loaded with Krebs-Henseleit buffer. (B Lower) EPR spectrum of nitrosylated myoglobin formed in a postischemic reperfused myoglobin^+/+ heart loaded with 50 μM [¹⁵N]nitrite. (C) Graph of the time courses of cardiac NO-heme levels in hearts loaded with 50 μM [¹⁵N]nitrite before, during, and after ischemia (n = 3). The levels of NO-heme complexes formed in myoglobin^+/+ hearts were quite high after 30 min of ischemia and 5 min of reperfusion compared with the concentrations of NO-heme in myoglobin^−/− hearts. (D) EPR spectra of the Mb[¹⁵N]NO complexes formed in nitrite-treated myoglobin^+/+ and myoglobin^−/− hearts (50 μM) before, during, and after global ischemia. The highest level of MbNO formation was observed 5 min after reperfusion.

Fig. 3.

Myoglobin is required for nitrite dependent inhibition of mitochondrial respiration. Representative oxygen electrode traces for myoglobin wild-type (A) and knockout (B) heart homogenates with either no treatment (0 μM) or treatment with cyanide (50 μM), or nitrite (5–25 μM). (C) Quantitation of several traces similar to A and B, where cyanide is 100% inhibition and 0 μM is 0% inhibition. (P < 0.01 vs. wild type; n = 5). **, P < 0.01.

Fig. 4.

Myoglobin-dependent nitrite-induced decrease of oxidative damage in myoglobin wild-type hearts compared to knockout hearts after ex vivo ischemia-reperfusion injury. (A) Cardiac NO-heme levels in myoglobin^+/+ and myoglobin^−/− hearts after 30 min of local ischemia and 5 min of reperfusion (n = 5). Untreated myoglobin^+/+ and myoglobin^−/− hearts exhibited differences in NO-heme concentration (P < 0.05). Nitrite (0.5 μM) increased NO-heme in presence of myoglobin by 70% (P < 0.05). Myoglobin^−/− hearts did not differ between control and nitrite treatment (0%, P values not significant). (B) cGMP in nitrite-treated (50 μM) myoglobin^+/+ and myoglobin^−/− hearts before ischemia and after 30 min of global ischemia and 15 min of reperfusion (n = 3). A 9.5-fold increase was observed in myoglobin^+/+ hearts, whereas myoglobin^−/− hearts displayed values below the detectable level. (C) In myoglobin^+/+ hearts, nitrite (0.5 μM) decreased the H₂O₂ production by 24% (P < 0.05), whereas in myoglobin^−/− hearts, nitrite increased the H₂O₂ generation by 22% (P < 0.05). (D) The aconitase level in myoglobin^+/+ hearts increased after nitrite treatment (0.5 μM) by 171%, whereas myoglobin^−/− hearts exhibited a decreasing aconitase level of 70% (P < 0.01). *, P < 0.05; **, P < 0.01.

Fig. 5.

Effect of myoglobin-dependent reduction of nitrite on the size of myocardial infarcts produced by left anterior coronary artery (LCA) occlusion in vivo. (A) Experimental protocol. (B) Representative 2,3,5-triphenyltetrazolium chloride stained sections showing viable (red) and ischemic (white) heart tissue. Evans blue dye (blue) delineates tissue outside the territory of the LCA and, therefore, not at risk for infarction from LCA occlusion. (C) Myocardial infarct size per area at risk (AAR). In myoglobin wild-type (^+/+) mice nitrite (48 nM) significantly reduced myocardial infarct size by 61% (P ≤ 0.01) in comparison to untreated myoglobin^+/+ and myoglobin knockout (^−/−) hearts, which did not differ among themselves (P values not significant). (D) Myocardial AAR per left ventricle (LV). The myocardial AAR per LV was similar within and between myoglobin^+/+ and myoglobin^−/− heart groups (P values not significant). **, P ≤ 0.01.