Mitochondrial oxidant stress triggers cell death in simulated ischemia–reperfusion

Abstract

To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O2 during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-XL in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-XL, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R.

β-Asarone protects PC12 cells against OGD/R-induced injury via attenuating Beclin-1-dependent autophagy

“…MMP is a sensitive indicator for evaluation of mitochondrial function. When mitochondrial membrane is damaged due to hypoxia, MMP decreases, which leads to ATP deficit and engulfment of damaged mitochondrial by the autophagosomes [16,24] . ATP deficit during ischemia and reperfusion leads to damage of energy-dependent ion pump on cellular membrane, concentration disorder of intracellular and extracellular ion, depolarization, and overload of intracellular Ca 2+ which in turn leads to a decrease of MMP [25] .…”

“…Intracellular free calcium concentration ([Ca 2+ ] i ) and mitochondrial membrane potential (MMP) are important indicators to reflect cell injury. It is reported that autophagy can be induced by increased [Ca 2+ ] i and decreased MMP [15,16] . Beclin-1, an autophagy related gene, is an essential indicator of autophagy.…”

The Protective Effect of Epigallocatechin-3 Gallate on Ischemia/Reperfusion Injury in Isolated Rat Hearts: Anex vivoApproach

“…Although ROS are produced by several extracellular and intracellular processes, in cardiomyocytes the mitochondria are the most relevant site for ROS formation [26]. Similarly, I/R-induced death is attenuated by over-expression of Mn-SOD or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by Cu, Zn-SOD, suggesting that mitochondrial ROS are one of the main causes of myocardial death.…”

“…Mitochondrial ROS and its associated mitochondrial damage is a determining factor in the loss of cardiomyocyte function and viability [26]. Similarly, overexpression of Mn-SOD, mitochondria-localized O2 − inhibitor, in mice protects against I/R injury in vivo [27].…”

Myocardial and Renal Ischemia/Reperfusion Injury and Experimental Models: Review

“…14,[17][18][19] Dahası, hücre içi Ca 2+ artışı, hücresel pH değerinin normalleşmesi ve serbest radikallerin reperfüzyon süresince aşırı üretimi mitokondriyal permeabilite geçiş porları [mitochondrial permeability transition pores; (mPTPs)]'nın açık kalma süresini uzatarak mitokondri içeriğinin sitozole geçmesine ve mitokondriyal çöküşe neden olur; bu durum ise hücre canlılığının kaybolmasıyla sonuçlanır. 4,[20][21][22][23] ROT, yüksek derişimdeki Ca 2+ ve oksidan kimyasallar mitokondriyal permeabilite geçişini [mitochondrial permeability transition; (MPT)]'yi indüklerken; magnezyum iyonu, düşük pH değeri ve siklosporin A engeller. Karaciğer, böbrek ve diğer hücrelerin hücre ölümüne karşı glisinin koruyucu etkisinin olduğu yapılan birçok hipoksi ve ATP tüketme modellerinde kanıtlanmıştır.…”