0432 : HL-1 cells: a cellular model to investigate cardioprotective mechanisms in ischemia/reperfusion context

Background and aims Pharmacological pre- and post-conditioningstrategies are among the more promising approaches to reduce ischemia/reperfusion- induced myocardial lesions but until now their molecular mechanisms are not completely described. However, this is a prerequisite to elaborate highly effective therapies. Before testing such therapeutic strategies on pre-clinical models and in humans, it is essential to have simple models, such as cell lines, to describe the molecular mechanisms involved in cardioprotection. The aim of our study was to develop experimental conditions to reproduce in vitro myocardial ischemia-reperfusion injuries. Methods and results we selected the HL-1 cell line, derived from a mouse atrial cardiomyocyte tumor lineage. To mimic myocardial ischemia-reperfusion lesions, cells were placed in a hypoxic chamber (about 0.1% O2) for different periods (4-22h). Evaluation of injuries was assessed by LDH release into culture supernatants. Lesions were observed from 10 h of hypoxia and gradually increased until reaching a maximum at 20 h. When cells were exposed to a 15 h period of hypoxia followed by reoxygenation (21% O2), we were unable to detect any further damage. Necrosis was mainly responsible for hypoxia-induced cell death with no obvious role of apoptosis. We also evidenced some alterations in mitochondrial functions using specific fluorescent probes: using TMRE and MitoSox, we showed a loss of mitochondrial membrane potential and an increased in ROS production, respectively. Using a Clark electrode, we also observed after 15 h of hypoxia, a significant reduction of mitochondrial oxygen consumption using different substrates. Conclusion We establish experimental conditions to mimic in vitro ischemia-reperfusion lesions. This model will enable us to test potential pharmacological pre-conditioningmolecules and to describe the mechanisms involved. The author hereby declares no conflict of interest