Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion

Abstract

Aims: Nitrated fatty acids (NO(2)-FA) have been identified as endogenous anti-inflammatory signalling mediators generated by oxidative inflammatory reactions. Herein the in vivo generation of nitro-oleic acid (OA-NO(2)) and nitro-linoleic acid (LNO(2)) was measured in a murine model of myocardial ischaemia and reperfusion (I/R) and the effect of exogenous administration of OA-NO(2) on I/R injury was evaluated.

Methods and results: In C57/BL6 mice subjected to 30 min of coronary artery ligation, endogenous OA-NO(2) and LNO(2) formation was observed after 30 min of reperfusion, whereas no NO(2)-FA were detected in sham-operated mice and mice with myocardial infarction without reperfusion. Exogenous administration of 20 nmol/g body weight OA-NO(2) during the ischaemic episode induced profound protection against I/R injury with a 46% reduction in infarct size (normalized to area at risk) and a marked preservation of left ventricular function as assessed by transthoracic echocardiography, compared with vehicle-treated mice. Administration of OA-NO(2) inhibited activation of the p65 subunit of nuclear factor kappaB (NFkappaB) in I/R tissue. Experiments using the NFkappaB inhibitor pyrrolidinedithiocarbamate also support that protection lent by OA-NO(2) was in part mediated by inhibition of NFkappaB. OA-NO(2) inhibition of NFkappaB activation was accompanied by suppression of downstream intercellular adhesion molecule 1 and monocyte chemotactic protein 1 expression, neutrophil infiltration, and myocyte apoptosis.

Conclusion: This study reveals the de novo generation of fatty acid nitration products in vivo and reveals the anti-inflammatory and potential therapeutic actions of OA-NO(2) in myocardial I/R injury.

Scheme 1

Experimental protocol for the assessment of OA-NO₂ effects in the murine I/R model.

Figure 1

Endogenous generation of OA-NO₂ following ischaemia and reperfusion. (A) Tissue concentration of OA-NO₂ in hearts from mice undergoing 30 min of ischaemia and 30 min of reperfusion, ischaemia without reperfusion or sham surgery. (B–D) Characterization of OA-NO₂ in heart tissue from mice subjected to I/R injury. The chromatograms show co-elution of OA-NO₂ extracted from heart tissue with synthetic OA-NO₂ (C) and the internal standard (D). (E) The table shows the peak area ratios corresponding to the two main products of OA-NO₂ collision induced dissociation: m/z 326/279 (neutral loss of HNO₂) and m/z 326/46 (formation of NO₂⁻), further confirming the identity of OA-NO₂ extracted from the heart tissue.

Figure 2

Endogenous generation of LNO₂ following ischaemia and reperfusion. (A) Tissue concentration of LNO₂ after I/R, ischaemia without reperfusion or sham surgery. (B) Chromatogram of LNO₂ extracted from mouse hearts subjected to ischaemia and reperfusion. LNO₂ eluted later than the [¹³C]-labelled cis-LNO₂ which was used as internal standard (C) and co-eluted with synthetic trans-LNO₂ (D). The slight shift in retention times between chromatograms (B and D) is due to variations between separate chromatographic runs.

Figure 3

LNO₂ is further oxidized to keto (A) and hydroxyl (B) derivatives in murine heart tissue after I/R; and 3 µM [¹³C]LNO₂ subjected to air oxidation overnight in Tris buffer (pH 8) was used as the precursor for oxidized ¹³C-labelled LNO₂ derivatives. These species served as internal standards for identification of endogenously produced oxidized derivatives of LNO₂ (C and D).

Figure 4

Effect of OA-NO₂ on infarct size and left ventricular function following ischaemia and reperfusion. (A) Representative images of hearts from vehicle- or OA-NO₂-treated animals stained with Evan's blue dye. (B) OA-NO₂ treatment at reperfusion, 15 min or 3 days prior to reperfusion lead to a significant reduction in infarct area/area at risk ratio when compared with vehicle-treated animals (*P < 0.01 vs. vehicle). Pretreatment with pyrrolidinedithiocarbamate (PDTC) abolished the effect of OA-NO₂. (C) Infarct area to left ventricular (IA/LV) areas reflected the results observed for the IA/AAR ratio (*P < 0.01 vs. vehicle). (D) No difference in AAR/LV ratio was observed between the different treatment groups. (E) OA-NO₂ treatment resulted in a significantly improved left ventricular function after I/R, as assessed by transthoracic M-mode echocardiography (*P < 0.01).

Figure 5

Inhibition of NFκB activation by OA-NO₂. (A) Reduction of NFκB p65 activity in myocardial tissue of OA-NO₂ treated animals following I/R. (B) Assessment of nitroalkylation of p65 in myocardial tissue.

Figure 6

Effect of OA-NO₂ on mRNA levels of adhesion molecules and MCP-1. (A) Reduced NFκB activation in OA-NO₂-treated animals was accompanied by a significant reduction of ICAM-1 mRNA expression. (B) Significant suppression of MCP-1 mRNA levels in OA-NO₂-treated animals. (C) VCAM-1 mRNA levels also showed a trend towards a reduction. All values are normalized to β₂-microglobulin.

Figure 7

Effect of OA-NO₂ on leukocyte infiltration and systemic inflammation following ischaemia and reperfusion. (A) Immunofluorescent staining for ICAM-1 (red) and CD31 (green) in vessels of OA-NO₂ (right panel) and vehicle-treated animals (left panel). OA-NO₂-treated animals showed a marked reduction in ICAM-1 immunoreactivity. (B) Neutrophil accumulation in infarcted zone of myocardium. Immunofluorescent staining for neutrophil-specific Ly-6-G revealed increased neutrophil infiltration in vehicle-treated mice. (C) Determination of MPO tissue levels confirmed increased neutrophil infiltration in vehicle-treated animals. (D) Serum TNFα levels were significantly reduced in OA-NO₂ treated animals 6 h after reperfusion. (E) Serum IL-6 levels underwent significant reduction in OA-NO₂-treated mice.

Figure 8

OA-NO₂ reduced apoptosis in myocardial tissue after ischaemia and reperfusion. (A) Immunofluorescent staining for TUNEL-positive cells. (B) Quantification of TUNEL-positive cells; *P < 0.001. (C) Assessment of caspase 3 activity. *P < 0.05 for comparison with sham; #P < 0.05 for comparison with OA-NO₂. No statistically significant difference was observed between OA-NO₂ treated animals and sham animals.