The role of hypoxia-inducible factor-1α and vascular endothelial growth factor in late-phase preconditioning with xenon, isoflurane and levosimendan in rat cardiomyocytes

The protective effects of late-phase preconditioning can be triggered by several stimuli. Unfortunately, the transfer from bench to bedside still represents a challenge, as concomitant medication or diseases influence the complex signalling pathways involved. In an established model of primary neonatal rat cardiomyocytes, we analysed the cardioprotective effects of three different stimulating pharmaceuticals of clinical relevance. The effect of additional beta-blocker treatment was studied as these were previously shown to negatively influence preconditioning. Twenty-four hours prior to hypoxia, cells pre-treated with or without metoprolol (0.55 mu g/ml) were preconditioned with isoflurane, levosimendan or xenon. The influences of these stimuli on hypoxia-inducible factor-1 alpha (HIF-1 alpha), vascular endothelial growth factor (VEGF) as well as inducible and endothelial nitric synthase (iNOS/eNOS) and cyclooxygenase-2 (COX-2) were analysed by polymerase chain reaction and western blotting. The preconditioning was proved by trypan blue cell counts following 5 h of hypoxia and confirmed by fluorescence staining. Five hours of hypoxia reduced cell survival in unpreconditioned control cells to 44 +/- 4%. Surviving cell count was significantly higher in cells preconditioned either by 2 x 15 min isoflurane (70 +/- 16%; P = 0.005) or by xenon (59 +/- 8%; P = 0.049). Xenon-preconditioned cells showed a significantly elevated content of VEGF (0.025 +/- 0.010 IDV [integrated density values when compared with GAPDH] vs 0.003 +/- 0.006 IDV in controls; P = 0.0003). The protein expression of HIF-1 alpha was increased both by levosimendan (0.563 +/- 0.175 IDV vs 0.142 +/- 0.042 IDV in controls; P = 0.0289) and by xenon (0.868 +/- 0.222 IDV; P < 0.0001) pretreatment. A significant elevation of mRNA expression of iNOS was measureable following preconditioning by xenon but not by the other chosen stimuli. eNOS mRNA expression was found to be suppressed by beta-blocker treatment for all stimuli. In our model, independently of the chosen stimulus, beta-blocker treatment had no significant effect on cell survival. We found that the stimulation of late-phase preconditioning involves several distinct pathways that are variably addressed by the different stimuli. In contrast to isoflurane treatment, xenon-induced preconditioning does not lead to an increase in COX-2 gene transcription but to a significant increase in HIF-1 alpha and subsequently VEGF.