Nitric Oxide synthase (NOS) does not contribute to simulated ischaemic preconditioning in an isolated rat cardiomyocyte model.

Abstract

It is widely accepted that nitric oxide (NO) is a trigger and mediator of late ischaemic preconditioning (IP), however its role in classic (protection observed within 2-4 hours after the IP stimulus) IP is less certain. In addition, the contribution of cardiomyocyte nitric oxide synthase (NOS) activation to NO production in ischaemia is unknown. The aim of this study was therefore to investigate the role of NOS, NO, reactive oxygen species (ROS) and cGMP in IP in an isolated cardiomyocyte model. Adult rat cardiomyocytes were isolated by collagenase perfusion. Hypoxia was induced by covering pelleted cardiomyocytes with mineral oil. The IP protocol was one 10 min hypoxia/20 min reoxygenation cycle, followed by 2 hr sustained hypoxia. Non-IP cells were subjected to 2 hr sustained hypoxia only. The contribution of NO was investigated by NOS inhibition (L-NAME 50 microM) or by pre-treatment of cells with a NO donor (SNP 100 microM), and that of ROS by inclusion of ROS scavengers (MPG and N-acetyl-cysteine) or pre-treatment with H(2)O(2). End-points were cellular cGMP content and cell viability as assessed by trypan blue exclusion (TBE) and cell morphology. IP significantly improved myocyte viability (54% increase in TBE) at the end of sustained hypoxia. Treatment of cells with L-NAME and ROS scavengers during either the IP protocol or during sustained hypoxia had no effect on cell viability after 2 hr hypoxia, whereas viability of non-IP cells treated with L-NAME during sustained hypoxia improved significantly. cGMP levels were reduced in IP cells. Pre-treatment with SNP and H(2)O(2) did not mimic IP. IP conferred cardioprotection in isolated cardiomyocytes. Protection in this model was not due to activation of cardiomyocyte NOS or ROS production. However, NOS activation induced by sustained hypoxia, appeared to be harmful to non-IP cells.