Abstract 3299: Endogenous Generation and Protective Effects of Nitro-fatty Acids in a Murine Model of Focal Cardiac Ischemia and Reperfusion
Aims: Nitro-fatty acids (NO2-FA) have been identified as endogenous anti-inflammatory signaling mediators generated by oxidative inflammatory reactions. Herein the endogenous generation of nitro-oleic acid (OA-NO2) and nitro-linoleic acid (LNO2) was measured in a murine model of myocardial ischemia and reperfusion (I/R), and the effect of exogenous administration of OA-NO2 on I/R injury was evaluated
Methods and Results: In C57/BL6 mice subjected to 30 min of coronary artery ligation, OA-NO2 and LNO2 generation was observed after 30 min of reperfusion, whereas no NO2-FA were detected in sham-operated mice and mice undergoing myocardial infarction without reperfusion. Intraperitoneal administration of 20 nmol/g body weight OA-NO2 during the ischemic episode significantly protected against I/R injury, with a 46% reduction in infarct volume (normalized to area at risk) and a marked preservation of left ventricular function as assessed by transthoracic echocardiography. Results from mice treated with the NFκB inhibitor pyrrolidinedithiocarbamate (PDTC), in concert with mass spectrometry-based identification of OA-NO2 adduction to the p65 subunit of NFκB in vivo supported that cardioprotection was lent by inhibition of NFκB. This was further corroborated by the OA-NO2 suppression of ICAM-1, MCP-1 and VCAM-1 expression, all being downstream mediators of NFκB. Additionally, inhibition of neutrophil infiltration and myocyte apoptosis by OA-NO2 further corroborated that OA-NO2 inhibits NFκB signaling. The mechanism of NO2-FA formation was explored in isolated mitochondria treated with the nitric oxide metabolite nitrite (500 μM) and linoleic acid (50 μM), wherein the formation of several nitro-linoleate regioisomers were detected in less than 5 min under physiologically-relevant I/R conditions. This included both cis and trans isomers of nitro-linoleic acid, as well as hydroxy and hydroperoxy nitro-linoleic acid derivatives, with all species displaying electrophilic reactivity.
Conclusions: This study shows for the first time the de novo generation of fatty acid nitration products in vivo and reveals the anti-inflammatory and therapeutic actions induced by OA-NO2 supplementation during ongoing myocardial I/R injury.