Nitro-fatty acids (NO2-FA) are endogenous mediators generated by reactions of nitrogen dioxide with unsaturated fatty acids. They are electrophilic and signal by reversibly reacting with nucleophilic cysteine and histidine residues on proteins, thus altering protein function. NO2-FA mediate cardioprotection in in vivo models of ischemia-reperfusion (IR) through not fully defined mechanisms. Mitochondria play a central role in both IR injury and redox signaling, with respiratory inhibition a common pathway in cardioprotective signaling. It was hypothesized that NO2-FA induce tissue-protective metabolic shifts through mitochondrial interactions.
Respirometry in isolated mitochondria demonstrated that complex II-linked, but not complex I-linked, respiration was inhibited by nitro-oleic acid (OANO2). Activity assays showed that inhibition of complex II was pH-dependent and reversible by the low molecular weight thiol β-mercaptoethanol (BME). Modification of the Fp subunit of complex II was confirmed following electrophoresis of mitochondrial proteins from OANO2-treated lysates: addition of BME displaced protein-bound OANO2 and led to the formation of BME-OANO2 adducts as measured by liquid chromatography/mass spectrometry (LC/MS). Extracellular flux analysis, where O2 consumption and media acidification are measured as surrogates for respiratory and glycolytic activity, was employed to determine the effects of OANO2 on bioenergetics in cardiomyoblasts. Pre-incubation with OANO2 inhibited basal and maximal respiration, while acute OANO2 injection inhibited respiration and promoted glycolysis, a tissue-protective shift in IR. Protection against IR injury to the heart was observed in a Langendorff-perfused heart model as improved cardiac output recovery during reperfusion in the presence OANO2.
Additional studies monitored metabolism of OANO2 in cardiac tissue and cells. NO2-FA are metabolized by prostaglandin reductase-1 (PGR1) and β-oxidation to yield non-electrophilic nitroalkanes and shorter chain nitroalkenes, respectively. PGR1 is inhibited by indomethacin and -oxidation by etomoxir, so these compounds were used to inhibit OANO2 metabolism. Indomethacin treatment attenuated OANO2 reduction and enhanced protein adduction by OANO2 and its electrophilic metabolites, while etomoxir favored the formation of non-electrophilic nitroalkanes and reduced β-oxidation products. Indomethacin also significantly enhanced OANO2 signaling in cardiomyoblasts, elevating heme oxygenase-1 expression compared to OANO2 alone. This work identifies mitochondrial actions of NO2-FA relevant to cardioprotection, and illuminates NO2-FA metabolic pathways relevant to signaling in the myocardium
