The role of fatty acyl CoA synthetase (FACS) in ischemia/reperfusion (I/R) injury has not been well established. Our earlier studies showed that triacsin C, a selective FACS inhibitor, decreases endothelial nitric oxide synthase (eNOS) palmitoylation and increases nitric oxide (NO) in cultured human coronary endothelial cells. In the present study, we tested the hypothesis that triacsin C would reduce infarct size and improve post-reperfusion cardiac function by increasing vascular NO. In isolated rat hearts, triacsin C, given during the first 5 minutes of reperfusion, significantly reduced infarct size and attenuated cardiac dysfunction during reperfusion. N G -nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor, 50 µM) completely abolished the protective effects of triacsin C. In the ischemic hind limb model, triacsin C significantly increased intravascular NO concentration during reperfusion, an effect that was blocked by L-NAME or S-methyl-L-thiocitrulline (SMTC, a selective neuronal NOS inhibitor), but not by 1400W (a highly selective iNOS inhibitor). Lastly, triacsin C significantly reduced L-NAME induced leukocyte rolling, adhesion, and transmigration in rat mesenteric circulation, as measured by intravital microscopy. In summary, this study provides novel evidence showing that triacsin C reduces myocardial infarct size, attenuates loss of post-reperfusion cardiac function, increases intravascular NO concentration and inhibits leukocyte recruitment. These pharmacologic properties suggest that triacsin C may be useful as an adjunct to Am.

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.

Endothelial dysfunction is characterized by decreased vascular NO availability. Elevated NEFA decreases eNOS activity. In cultured cells, we found that fatty acyl CoA synthase (FACS) inhibitor Triacsin C (TC) interrupted eNOS palmitoylation, increasing eNOS activity but not changing vascular‐active eicosanoids. Hypothesis: TC mitigates endothelial dysfunction by increasing NO and decreasing NEFA. In this study, intravascular NO synthesis was measured by electrochemistry in the ischemic hind limb with heparinized rat model in a time course design. The post‐ischemic NO was significantly elevated in TC (100 μg/kg)‐treated animals than controls. NOS inhibitor treatments in this model implicate a role of eNOS, but not iNOS. Acute hyperlipidemia was induced by a bolus dose of Intralipid®(1 ml) to increase NEFA. Pre‐treatment with TC had no effect on total plasma lipids or triglycerides, either before or after Intralipid®. TC reduced baseline plasma NEFA from 240 ± 26.8 to 147 ± 14.3 μg/ml (p=0.038). Fifteen minutes after Intralipid®, NEFA rose to 1461 ± 130 μg/ml (control), which was blunted by TC to 393 ± 2.55 μg/ml (p=0.0079). By 85 min., the difference had subsided (194 ± 10.2 control vs. 281 ± 74.8 μg/ml TC; p=0.74). The TC effect was not uniform across all fatty acid species measured. These data show that TC increases post‐ischemic eNOS activity and blunted plasma NEFA induced by acute hyperlipidemia.

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