Artemisinin Protects The Myocardium From Ischaemia Reperfusion Injury Via PI3K/AKT/iNOS Cell Survival Pathway

M.A Babba1, O Janneh2, H.L Maddock1, A Hussain1. 1Coventry University,Coventry, Coventry, UK, 2St George\'s,University of London, London, UK

Introduction Artemisinin (ART) exhibits potent anti-malarial, anti-tumour and anti-inflammatory activities. Artemisinin has been shown to limit injury in the ischaemic reperfused myocardium although the associated intracellular signalling pathways associated remain to be elucidated. The aim of the current study was to determine involvement of the PI3K/AKT/iNOS cell survival pathway in Artemisinin mediated cardioprotection.

Methods. Isolated perfused rat hearts or primary adult rat cardiac myocytes were subjected to ischaemia/reperfusion or Hypoxia/Reoxygenation, respectively. ART (4.3 µM) was administered at the onset of reperfusion/reoxygenation in the presence and absence of PI3K inhibitor Wortmannin (100nM), the non-selective nitric oxide inhibitor L-NAME (100 µM) or the inducible nitric oxide (iNOS) inhibitor Aminoguanidine (100 µM). Hearts underwent triphenyl-tetrazolium staining for infarct size assessment or were frozen for Western blot analysis for p-AKT. Cardiac myocytes were assessed for myocyte viability via MTT assay and fluorimetric analysis for cleaved-caspase 3, iNOS and p-eNOS.

Results. ART significantly reduced infarct size compared to non-treated controls (38±0.03% IR+ART vs 50±0.04% IR, p < 0.001). Co-administration with Wortmannin, Aminoguanidine or L-NAME abolished the infarct sparing effects (44±1.47% IR+ART+W) vs. 38±2.55% IR+ART, p<0.001), (48 ±1% IR+ART+A) and (47± 1% IR+ART+L, p< 0.01 ). ART showed improvement in myocyte viability (66 ± 6% H/R + ART vs. 29 ± 6% H/R, p<0.001) and decreased cleaved-caspase 3 activity compared to H/R control (17 ± 2% H/R +ART vs. 27 ± 2% H/R, p<0.001).The cytoprotective effect was abolished by the co-administration of Wortmannin, Aminoguanidine or L-NAME. Art dependent cytoprotection was associated with upregulation of p-Akt, iNOS and p-eNOS levels compared to time matched controls that was abolished by co-administration of either Wortmannin, Aminoguanidine or L-NAME.

Conclusions. This is the first study to show Artemisinin limits myocardial injury via recruitment of the PI3K/AKT/iNOS cell survival pathway.