Cell death in hyppxic injury : signaling mechanisms and dynamics in the decision making process

[摘要] ENGLISH ABSTRACT: Three main morphologies of cell death have been described in the diseasedmyocardium, type I, better known as apoptotic cell death, which is characterized bycell shrinkage and chromatin condensation, type II, or cell death with autophagy,presents a morphology with intracellular accumulation of autophagic vacuoles andtype III, better known as necrosis, is characterized by cellular swelling and rapidloss in cellular membrane integrity. However, recent literature strongly arguesagainst rigid classifications in the context of cell death mechanisms but rathersuggests to adopt a view of cell death as a dynamic and integrative cellularresponse. Furthermore, the contribution of autophagy in cell death or cell survivalis still poorly understood. Therefore the aims of this study were twofold: (i) tocharacterize the contribution of each cell death type in context of the severity andduration of an ischaemic insult and (ii) to determine whether manipulation of theautophagic pathway affects the contribution of cell death and translates intoprotection of the heart.Rodent derived cardiac myoblast cells were grown in Dulbecco's Modified Eagle'sMedium (DMEM) supplemented with 10% fetal bovine serum (FBS), and incubatedunder 5% CO2 conditions. Cells were submitted to protocols of 2, 4 and 8 hrs ofsimulated ischaemia (SI) under hypoxic conditions in a humidified environmentcontaining 0.1% O2, 5% CO2 and the balance N2, followed by 1 hr of reperfusionrespectively. We employed a modified ischaemic buffer containing either 2-deoxy-D-glucose, sodium dithionate or both, with the aim to create an ischaemic insult ofmild (mild SI), moderate (moderate SI) and severe (severe SI) characterrespectively.We evaluated the contribution of each cell death mode using a combination ofviability- and ATP assays. Molecular markers for each cell death process such asLC3, PARP and HMGB1 were evaluated using 3-dimensional fluorescencetechniques as well as western blot analysis and flow cytometry. Next, autophagy was induced or inhibited prior to the ischaemic insult, using rapamycin and 3MArespectively, and similar parameters were evaluated after 2 hours of mild ormoderate SI. Propidium Iodide exclusion and Fluorescence Resonance EnergyTransfer (FRET) in combination with mitochondrial inner membrane depolarizationwere employed to assess the onset of cell death dynamically. Flow cytometry wasemployed to evaluate the degree of protection. In addition, the ATP levels andreactive oxygen species (ROS) were evaluated.Our results strongly indicate a differential induction of cell death, which isdependent on the severity and duration of the ischaemic insult. Mild SI led to theinduction of autophagy and apoptosis, whilst moderate or severe SI induced bothapoptotic and necrotic cell death without an indication of autophagy. Only mild SI,but not moderate and severe SI, resulted in an ATP surge.Moreover, our data provide direct evidence that increased autophagy delays theloss of cellular membrane integrity and delays caspase-3 activation as well asmitochondrial depolarization in ischaemic cardiomyocytes. Our results show aprofound effect of increased autophagy on the onset of apoptosis as well asnecrosis under simulated ischaemic conditions, providing cellular protection. ThisATP surge observed during mild SI was abolished with increased autophagy.Furthermore, our results indicate a profound effect of autophagy on ROSgeneration. Under normoxic conditions, increased autophagy induced a significantdecrease in ROS while the inhibition of autophagy significantly increased ROSgeneration. However, when increasing or decreasing autophagy prior to theischaemic insult, ROS increased significantly in both scenarios.The results suggest that the severity of ischaemia determines the mode of celldeath differentially. An increase in autophagic responsiveness and flux, as inducedthrough rapamycin treatment, provides a selective advantage for tissue againstinjury, possibly by maintaining intracellular ATP levels through the provision ofmetabolic substrates. Autophagy is described as an inherent cellular mechanism vwhich affects the onset of cell death and exhibits protective effects in the ischaemicmyocardium when upregulated prior to the ischaemic insult.The protective effect of increased autophagy was mirrored in the isolated perfusedrat heart model, reflected by improved functional recovery duringischaemia/reperfusion.

[发布机构] Stellenbosch University