Brief periods of nitric oxide inhalation protect against myocardial ischemia-reperfusion injury

Abstract

Background: Prolonged breathing of nitric oxide reduces myocardial ischemia-reperfusion injury, but the precise mechanisms responsible for the cardioprotective effects of inhaled nitric oxide are incompletely understood.

Methods: The authors investigated the fate of inhaled nitric oxide (80 parts per million) in mice and quantified the formation of nitric oxide metabolites in blood and tissues. The authors tested whether the accumulation of nitric oxide metabolites correlated with the ability of inhaled nitric oxide to protect against cardiac ischemia-reperfusion injury.

Results: Mice absorbed nitric oxide in a nearly linear fashion (0.19 +/- 0.02 micromol/g x h). Breathing nitric oxide rapidly increased a broad spectrum of nitric oxide metabolites. Levels of erythrocytic S-nitrosothiols, N-nitrosamines, and nitrosyl-hemes increased dramatically within 30 s of commencing nitric oxide inhalation. Marked increases of lung S-nitrosothiol and liver N-nitrosamine levels were measured, as well as elevated cardiac and brain nitric oxide metabolite levels. Breathing low oxygen concentrations potentiated the ability of inhaled nitric oxide to increase cardiac nitric oxide metabolite levels. Concentrations of each nitric oxide metabolite, except nitrate, rapidly reached a plateau and were similar after 5 and 60 min. In a murine cardiac ischemia-reperfusion injury model, breathing nitric oxide for either 5 or 60 min before reperfusion decreased myocardial infarction size as a fraction of myocardial area at risk by 31% or 32%, respectively.

Conclusions: Breathing nitric oxide leads to the rapid accumulation of a variety of nitric oxide metabolites in blood and tissues, contributing to the ability of brief periods of nitric oxide inhalation to provide cardioprotection against ischemia-reperfusion injury. The nitric oxide metabolite concentrations achieved in a target tissue may be more important than the absolute amounts of nitric oxide absorbed.

Conflict of interest statement

Conflict of Interest Statement: The authors, Drs. Zapol and Bloch, have obtained patents relating to the use of inhaled nitric oxide. These patents are assigned to Massachusetts General Hospital (Boston, Massachusetts), which has licensed them to IKARIA (Clinton, New Jersey) and Linde Gas Therapeutics (Lidingo, Sweden). Dr. Zapol receives royalties and Dr. Bloch has received grants from IKARIA and Linde, which helped us to study inhaled nitric oxide.

Figures

Figure 1

The absorption of nitric oxide by mice breathing 80 ppm nitric oxide (ppm NO) for 60 min. Mice breathed air supplemented with 80 ppm NO for 60 min in a vented chamber (n=4). Nitric oxide concentrations were recorded continuously at the chamber outlet during the first 30 sec, and thereafter, inlet and outlet were sampled every 15 sec. The difference between inlet and outlet concentrations multiplied by the gas flow rate and time provides an accurate measurement of total nitric oxide gas absorbed by each mouse. The line represents the mean absorption of nitric oxide, and SEM is shown at one minute intervals.

Figure 2

Distribution and kinetics of accumulation of NO-metabolites in mice breathing nitric oxide (plasma and erythrocytes). Concentrations of NO-metabolites were measured in blood of mice breathing air supplemented with nitric oxide for 0, 0.5, 5, 15, and 60 min (n=5-7). Abbreviations: erythrocytes (RBC), nitrosyl-heme species (NO-heme), N-nitrosamines (RNNO), S-nitrosothiols (RSNO).*Phttp://www.anesthesiology.org. - Web Enhancement #2-Table 1.

Figure 3

Distribution and kinetics of accumulation of NO-metabolites in mice breathing NO (heart, lung, brain and liver). Concentrations of NO-metabolites were measured in tissues of mice breathing air supplemented with nitric oxide for 0, 0.5, 5, 15, and 60 min (n=4-7). Abbreviations: nitrosyl-heme species (NO-heme), N-nitrosamines (RNNO), S-nitrosothiols (RSNO). Additional information regarding each concentration, the number of animals studied, and the P-value are available on the Anesthesiology Web site at http://www.anesthesiology.org. - Web Enhancement #3-Table 2.

Figure 4

Effects of hypoxia on the cardiac levels of NO-metabolites. Concentrations of cardiac NO-metabolites were measured in cardiac tissue of mice breathing air (Baseline), 8% oxygen (Hypoxia), air supplemented with nitric oxide (Normoxia+nitric oxide), or 8% oxygen balance nitrogen supplemented with nitric oxide (Hypoxia+nitric oxide) for 60 min. *P‡P<0.05 vs. Hypoxia, +P<0.05 vs. Normoxia+nitric oxide.

Figure 5

Measurement of nitrite (A) and nitrate (B) in urine from mice breathing nitric oxide. Mice received air supplemented with nitric oxide for 0, 5, and 60 min (n=8, 7, and 9, respectively). *P

Figure 6

Inhalation of nitric oxide for short durations limits myocardial ischemia-reperfusion injury. All mice underwent left coronary artery occlusion for 60 min followed by 24 h of reperfusion. Mice received nitric oxide during ischemia for 60 min (n=10 and 9 for control and nitric oxide inhaled mice, respectively, Panel A), 5 min immediately before reperfusion (n=9 and 8 for control and nitric oxide inhaled mice, respectively, Panel B), or 0.5 min immediately before reperfusion (n=7 and 6 for control and nitric oxide inhaled mice, respectively, Panel C). Control mice did not receive nitric oxide. *P<0.05 vs. control. Abbreviations: area at risk (AAR), left ventricle (LV), myocardial infarction (MI).