The Effect of IKK-16 on Infarct Size and Inflammation Following Myocardial Ischaemia/Reperfusion

Myocardial ischaemia and reperfusion results in the activation of nuclear factor-kappa B (NF-кB), and generation of NF-кB –dependent proteins. There is evidence that inhibition of NF-kB during myocardial ischaemia and reperfusion (MI) of the heart has beneficial effects (1). The aim of this project was to evaluate the effects of a specific inhibitor of IKK (IKK-16; (2))on infarct size and inflammation in a rat model of myocardial ischaemia and reperfusion.

Methods: Eighty-three male Wistar rats were anaesthetised (120 mg/kg sodium thiopentonei.p.) and catheters placed in the carotid artery and jugular veins. The rat was intubated and a full thoracotomy performed to expose the heart. The pericardium was carefully removed and a 6-0 suture placed around the left anterior descending (LAD) coronary artery. MI was induced by occlusion of the LAD 25 min followed by reperfusion for 2 h. At the end of the reperfusion period, the artery was re-occluded and 1 ml of 5% Evans blue dye solution was injected as i.v. bolus via the jugular artery. At the end of the experiment, animals were sacrificed by an overdose of sodium thiopentane and the heart excised and placed into cold saline. The left ventricle (LV) was isolated, and the area at risk quantified as a percentage of the LV. The ischaemic tissue was then stained with nitroblueteterozolium (NBT) 37˚C for 30 min to identify viable and non-viable tissue. Data was analysed by one-way ANOVA followed by Tukey’s post hoc test. A P value <0.05 was considered significant.

Results: When compared to surgery alone (sham, n=8), LAD-occlusion/reperfusion (MI-control, vehicle treatment, n=17) resulted in an increase of infarct size from 12.8 ± 2.3% to 44.6 ± 2.5% (P<0.05). Administration of IKK-16 (1 mg/kg i.v. bolus upon reperfusion) caused a significant reduction in infarct size to 33.6 ± 3.0% (n=12; P<0.05 when compared to control). In comparison (positive control) preconditioning of rats with two cycles of ischaemia (2 min) followed by reperfusion (5 min) caused a significant reduction in infarct size to from 44.6±2.5% (control, see above) to 31.8 ± 2.2% (n=6; P<0.05). When compared to sham, MI-rats treated with vehicle exhibited (in cardiac tissue obtained from the AAR) an increase in the nuclear translocation of p65 to the nucleus and, hence, activation of NF-кB. The degree of nuclear translocation of p65 was reduced in MI-rats treated with either IKK-16 upon reperfusion or subjected to ischaemic preconditioning prior to MI. When compared to sham, MI-rats treated with vehicle exhibited (in cardiac tissue obtained from the AAR) an increase in neutrophil accumulation (measured as myeloperoxidase MPO-activity), which was significantly reduced in MI-rats treated with ischaemic preconditioning, but not IKK-16 (n=4, P>0.05 v control).

Summary & Conclusion: Inhibition of the activation of NF-кB during reperfusion with IKK-16 resulted in a reduction of infarct size similar to the one afforded by ischaemic preconditioning. The mechanisms underlying the observed cardioprotective effects of IKK-16 are not clear, but are not related to prevention of the accumulation of neutrophils within the ischaemic myocardium.

(1)- Li, C., Browder, W., Kao, R.L.. (1999). Early activation of transcription factor NF-kB during ischemia in perfused rat heart. Am J Physiol. 276 (1), H543- H552.

(2)- Waelchli, R. et al., (2006). Design and preparation of 2-benzamido-pyrimidines as inhibitors of IKK. Bioorganic & Medicinal Chemistry Letters. 16 (1), 108-112.