Nitrite anion provides potent cytoprotective and antiapoptotic effects as adjunctive therapy to reperfusion for acute myocardial infarction

Abstract

Background: Accumulating evidence suggests that the ubiquitous anion nitrite (NO2-) is a physiological signaling molecule, with roles in intravascular endocrine nitric oxide transport, hypoxic vasodilation, signaling, and cytoprotection. Thus, nitrite could enhance the efficacy of reperfusion therapy for acute myocardial infarction. The specific aims of this study were (1) to assess the efficacy of nitrite in reducing necrosis and apoptosis in canine myocardial infarction and (2) to determine the relative role of nitrite versus chemical intermediates, such as S-nitrosothiols.

Methods and results: We evaluated infarct size, microvascular perfusion, and left ventricular function by histopathology, microspheres, and magnetic resonance imaging in 27 canines subjected to 120 minutes of coronary artery occlusion. This was a blinded, prospective study comparing a saline control group (n=9) with intravenous nitrite during the last 60 minutes of ischemia (n=9) and during the last 5 minutes of ischemia (n=9). In saline-treated control animals, 70+/-10% of the area at risk was infarcted compared with 23+/-5% in animals treated with a 60-minute nitrite infusion. Remarkably, a nitrite infusion in the last 5 minutes of ischemia also limited the extent of infarction (36+/-8% of area at risk). Nitrite improved microvascular perfusion, reduced apoptosis, and improved contractile function. S-Nitrosothiol and iron-nitrosyl-protein adducts did not accumulate in the 5-minute nitrite infusion, suggesting that nitrite is the bioactive intravascular nitric oxide species accounting for cardioprotection.

Conclusions: Nitrite has significant potential as adjunctive therapy to enhance the efficacy of reperfusion therapy for acute myocardial infarction.

Figures

Figure 1

Schematic diagram of experimental protocol and major measurements. MR imaging was performed at baseline, between approximately 30 and 50 minutes during the first hour of ischemia, was repeated with similar timing during the second hour of ischemia, and between 4 and 6 hours after reperfusion. Microspheres were injected after each MR scans except the baseline scan. Histopathology was performed at least 6 hours after reperfusion for optimal triphenyltetrazolium chloride (TTC) staining.

Figure 2

Nitrite, S-nitrosothiol (SNO), and nitrosyl-hemoglobin (RxNO) concentrations. Nitrite significantly increased (p

Figure 3

Area at risk was measured volumetrically based on the hypointense zone (red arrows) on the perfusion images from a single animal from the papillary muscle level to the apex (A). Infarct size was measured as the size of the pale zone on the TTC stained myocardium (B). The background was masked to better delineate the endocardial borders. For similar sized perfusion defects (C), both nitrite infusions resulted in small subendocardial infarcts while the saline group tended to have nearly transmural infarcts. The red arrows on the perfusion images (C) delineate the epicardial extent of the area at risk. The green arrows delineate corresponding points on the TTC stained myocardium. Note the TTC negative zone encompasses a much smaller percent of the area at risk in the nitrite treated animals.

Figure 4

Infarct size normalized to area at risk was significantly reduced by both the 60-minute nitrite infusion (p

Figure 5

Both nitrite infusions significantly reduced apoptosis relative to the saline control infusion, particularly in layers 3 and 4 (60-min p

Figure 6

Although variations in preload, afterload, and rate pressure product confound interpretation of myocardial salvage associated with the 60-min nitrite infusion, there were no significant differences in preload (inversely related to ischemic zone end diastolic wall thickness), afterload (as measured by systolic wall stress), or rate pressure product (RPP) when comparing saline control and the 5-min nitrite group. OC30 = 30 minutes into occlusion (ischemia); OC90 = 90 minutes into occlusion (ischemia).

Figure 7

Left ventricular ejection fraction (LVEF) decreased to similar extent in all three groups (p

Figure 8

Effects of nitrite on perfusion during ischemia and microvascular obstruction after reperfusion. Microsphere blood flow (panel A) and qualitative perfusion abnormalities on MRI (inset) remained severely abnormal during ischemia despite potential vasodilation related to nitrite. After reperfusion, there was less microvascular obstruction in the two nitrite treated groups (*) relative to control. This was visible on serial MR perfusion images as mild or patchy residual hypointense zones relative to the dark defects seen during ischemia (example shows a single slice at the three time points from one animal treated with nitrite).