Nitrosylheme Formation And Nitric Oxide Signaling During Brief Myocardial Ischemia

In addition to generation from specific nitric oxide (NO) synthases, we have observed that NO formation from nitrite occurs in the ischemic heart. While NO binding to heme-centers is the basis for NO-mediated signaling, as occurs through guanylate cyclase (GC), it was not known if this process is triggered with physiologically relevant periods of sublethal ischemia and if nitrite serves as a critical substrate. Therefore, EPR studies to measure nitrosyl-heme formation during the time course of myocardial ischemia and reperfusion and

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MAGNETIC FIELD (Gauss)

Fig. 8. EPR spectra of NO-heme formation in hearts during ischemia. Top to bottom, ischemic durations of 0-240 min. Spectra were recorded at 77 K with a frequency of 9.786 GHz; 10.0 mW power.

the role of nitrite in this process were performed [3]. Ischemic hearts loaded with nitrite 50 ^M showed prominent spectra consisting of six-coordinate nitrosyl-heme complexes, primarily NO-myoglobin, that increased as a function of ischemic duration. In nonischemic-controls these signals were not seen (Fig. 8). Quantitative analysis of EPR spectra showed that total NO-heme concentrations within the heart were 6.6 ± 0.7 ^M after 30 min of ischemia, and 12.7 ^M at 240 min. With the increasing nitrite-loading-dose concentrations a linear correlation was observed between dose and amount of Mb-NO formed from 3.04 ± 0.01 ^M in hearts loaded with 25 ^M nitrite to 27.0 ± 0.3 ^M in hearts with 400 ^M nitrite. NO-Mb in controls (without nitrite load) also progressively increased as a function of ischemic duration but was about 8-10-fold lower than the nitrite-loaded hearts with concentrations of 0.5 or 1.6 ^M after 30 or 240 min, respectively.

The levels of NO-heme complexes formed in ischemic myocardium are quite high compared with the concentrations of free NO in cells of 10-100 nM or less [68]. To prove that the observed NO-heme complex formation was derived from nitrite, isotope tracer experiments were performed measuring NO-heme formation in hearts infused with isotopically labeled 15N nitrite. With 15N nitrite labeling, the characteristic centrally split 15NO-Mb spectrum was seen, while with 14n nitrite the typical 14NO-Mb spectrum was observed [3]. Thus, under ischemic conditions myoglobin binds and stabilizes nitrite-derived NO in the form of NO-Mb complexes, and these complexes serve as a store of NO.

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Ischemia Reperfusion

Time (min)

Fig. 9. Time course of NO-heme levels before, during, and following ischemia. From EPR measurements on a series of three nitrite loaded hearts at each preischemic (PI), ischemic, and reperfusion time.

Upon reperfusion the concentration of NO-heme complexes decrease, as would be expected with the reoxygenation that accompanies reperfusion. Initially a rapid decrease was seen in the observed NO-heme EPR spectra over the first 15 min of reperfusion followed by a slow decrease thereafter, with 26% of the signal intensity persisting even after 45 min of reperfusion (Fig. 9). The time course of the early decrease parallels the oxidant burst accompanied by superoxide and superoxide-derived radical generation that occurs in ischemic myocardium. It is possible that the early rapid decrease in NO-heme complexes is due to superoxide or other radical reaction with the bound NO. The subsequent slow decrease could be due to a slow rate of spontaneous oxidation secondary to the levels of oxygen present in the reperfused heart. The decrease in NO-heme signal seen upon reperfusion could be due to oxidation or facilitated release/exchange of the NO bound at the heme site of Mb. With oxidation, nitrate formation would be observed, whereas for released NO, nitrite formation would be expected. In the hearts preloaded with nitrite, nitrite levels were elevated only over the first minute of reperfusion, which can also be explained by simple nitrite washout. However, nitrate levels remained elevated over the first 5 min of reperfusion and exhibited a time course of decrease that paralleled the observed decrease in NO-heme concentrations within the heart. As described above, the observed NO-heme formation is primarily due to the formation of NO-Mb complexes. However, trace amount of five coordinate NO-heme complexes were also seen, as would arise from NO bound to guanylate cyclase [69,70]. Although these could also arise from other heme centers [71,72].

NO binding to the heme of sGC (soluble guanylate cyclase) is the critical event triggering the activation of sGC that results in cyclic guanosine monophosphate (cGMP) formation in the presence of guanosine triphosphate (GTP). It has been previously demonstrated that NO-Mb effectively donates NO-heme to sGC, resulting in activation of the enzyme with cGMP formation [73]. In view of this, we performed experiments to determine whether the observed NO-Mb formation in the ischemic heart was associated with activation of sGC.

Fig. 10. Levels of cGMP in control and 50 |M nitrite treated hearts. Tissue cGMP levels were measured in normally perfused nonischemic hearts, after 30 min of 37°C global ischemia, or after reperfusion for 15 min following after 30 min of ischemia. Control untreated hearts (white bars) and nitrite (50 |iM)-treated hearts (black bars) were studied. Values are expressed in units of fmol/mg of heart tissue, wet weight.

Fig. 10. Levels of cGMP in control and 50 |M nitrite treated hearts. Tissue cGMP levels were measured in normally perfused nonischemic hearts, after 30 min of 37°C global ischemia, or after reperfusion for 15 min following after 30 min of ischemia. Control untreated hearts (white bars) and nitrite (50 |iM)-treated hearts (black bars) were studied. Values are expressed in units of fmol/mg of heart tissue, wet weight.

In nonischemic hearts that were either nitrite-treated or untreated, only low levels of cGMP were seen. However, after 30 min of ischemia, a modest increase in cGMP levels was present in untreated hearts, whereas a large 4-fold increase was seen in nitrite-treated hearts compared with preischemic levels. Furthermore, in ischemic hearts the levels of cGMP was 2.5-fold higher with nitrite treatment than in otherwise identical untreated control hearts (Fig. 10). Upon reperfusion the levels of cGMP declined, and after 15 min of reperfusion more than a 2.4-fold decrease in cGMP levels was observed from ischemic values. These changes in cGMP levels paralleled the changes seen for NO heme levels. This observed increase in cGMP levels was independent of NOS, since 1 min of pre-infusion with 1.0 mM W-G-monomethyl-L-arginine (NOS inhibitor) before the onset of ischemia did not significantly change the levels of cGMP (compared with 1.6 ± 0.3 fmol/mg wet weight for treated hearts compared with 2.0 ± 0.4 fmol/mg wet weight for hearts without NOS inhibition). Activation of sGC accompanied the formation of NO-Mb complexes and thus, the observed nitrite-derived NO-heme formation is paralleled by activation of myocardial-signaling pathways.

Nitrite-mediated NO-heme formation during ischemia is paralleled by subsequent depressed recovery of contractile function upon reperfusion. In nitrite-treated hearts, the recovery of contractile function as measured by left ventricular-developed pressure and rate-pressure product was significantly decreased, compared with that in untreated control hearts, with final recovery at 30 min diminished by more than 3-fold. In contrast to the impaired recovery of contractile function in nitrite-treated hearts, the recovery of coronary flow was paradoxically mild elevated.

Thus, nitrite-mediated nitrosyl heme formation occurs in ischemic myocardium and can be an important regulator of myocardial signaling and injury in the postischemic heart.

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