Autophagy is a major intracellular pathway for degrading damaged or dysfunctional cyctoplasmic aggregates, for the purposes of maintaining cellular homeostasis under normal and pathophysiological conditions. Studies have shown that autophagy may be induced by both extracellular and intracellular signals, including oxidative stress and serum starvation, where degradation may function as an adaptive process in order to protect against ischemia-reperfusion injury in the heart.
In this study, we hypothesize that exposing cultured vascular endothelial cells to simulated ischemia-reperfusion, or hypoxia-reoxygenation, will result in mitochondrial fission and thus the removal of damaged mitochondria by autophagy (mitophagy). We developed protocols to assess and quantify autophagy and mitophagy in ECs exposed to 1h, 4h, 8h of hypoxia and 1h and 4h of hypoxia with varying times of reoxygenation. We have also quantified markers of autophagy and mitophagy, protein light chain 3 (LC3-II) and Beclin, and have evaluated the morphology of mitochondria under 4h of hypoxia via fluorescence microscopy. Degradation of mitochondria at 4h of hypoxia was also evaluated by cytochdrome c oxidase subunit IV (COX IV) expression.
Results from immunoblotting and immunocytochemistry indicate that hypoxic conditions may lead to mitochondrial fission and the removal of dysfunctional mitochondria by autophagy. Our results demonstrate 1-4h as a key time point for EC survival, and that in ECs exposed to 1-4h hypoxia, there is a greater accumulation of autophagosomes (heightened LC3-II levels). COXIV expression showed the possibility for mitochondrial degradation and removal via through mitophagy.
In order to determine possible relationships with mitochondrial morphology (fission) and mitophagy, hypoxia at 4h also demonstrated heightened fragmentation of mitochondria, which was lessened in the presence of CsA. The effect of CsA decreasing fragmented mitochondria may link mitophagy to the mitochondrial depolarization in ECs. These discoveries may shed light on the possible molecular pathways that regulate cellular and mitochondrial homeostasis and protection in maintaining cellular quality control in the face of ischemia-reperfusion injury.