Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation

Abstract

. Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation. Am J Physiol Heart Circ Physiol 284: H549-H558, 2003. First published October 10, 2002 10.1152/ajpheart.00708.2002.-Ischemia-reperfusion injury induces cell death, but the responsible mechanisms are not understood. This study examined mitochondrial depolarization and cell death during ischemia and reperfusion. Contracting cardiomyocytes were subjected to 60-min ischemia followed by 3-h reperfusion. Mitochondrial membrane potential (⌬⌿m) was assessed with tetramethylrhodamine methyl ester. During ischemia, ⌬⌿m decreased to 24 Ϯ 5.5% of baseline, but no recovery was evident during reperfusion. Cell death assessed by Sytox Green was minimal during ischemia but averaged 66 Ϯ 7% after 3-h reperfusion. Cyclosporin A, an inhibitor of mitochondrial permeability transition, was not protective. However, pharmacological antioxidants attenuated the fall in ⌬⌿m during ischemia and cell death after reperfusion and decreased lipid peroxidation as assessed with C11-BODIPY. Cell death was also attenuated when residual O2 was scavenged from the perfusate, creating anoxic ischemia. These results suggested that reactive oxygen species (ROS) were important for the decrease in ⌬⌿m during ischemia. Finally, 143B-0 osteosarcoma cells lacking a mitochondrial electron transport chain failed to demonstrate a depletion of ⌬⌿m during ischemia and were significantly protected against cell death during reperfusion. Collectively, these studies identify a central role for mitochondrial ROS generation during ischemia in the mitochondrial depolarization and subsequent cell death induced by ischemia and reperfusion in this model. reactive oxygen species; hypoxia; oxidants REACTIVE OXYGEN SPECIES (ROS) have been implicated as participants in the myocardial damage induced by ischemia-reperfusion (I/R) (1,4,9,17,21,25). Most studies have focused on the importance of oxidant stress generated during reperfusion, when a burst of ROS is generated after oxygen is reintroduced into the system after a prolonged period of ischemia (32, 39). However, growing evidence suggests that oxidant stress begins during ischemia before reperfusion. For example, in cardiomyocytes subjected to simulated I/R, we observed (32, 33) an increase in ROS generation during ischemia followed by a large burst of oxidant production during the first few minutes after reoxygenation. In that model, antioxidants were more protective when given throughout the experiment than when given only at reperfusion, which supports the idea that oxidants generated during the ischemic phase contribute to cell injury and are important determinants of cell survival and recovery of function (2, 35).ROS generation cannot occur during ischemia unless some residual O 2 is still present. Previous studies using cardiomyocytes revealed that trace levels of O 2 are still detectable during simulated ischemia (PO 2 ϭ 5-7 mmHg). During ischemia, indexes of oxidant stress were attenuated by mitochondrial electron trans...

Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation

“…However, other cells may not exhibit such rapid and high sensitivity to hypoxia. For example, more severe and prolonged hypoxia treatments may be necessary to induce mitochondrial depolarization in cardiomyocytes (45). Notwithstanding, our results with dopamine-containing PC12 cells confirm the conclusions of previous studies in other systems that hypoxia depolarizes ⌬⌿ m , as demonstrated by using different voltage-sensitive dyes (44,45), and that hypoxia increases the overall cellular ROS level before the onset of reperfusion (6,31,46).…”

“…This mitochondrial sequence is present in many mammalian MSRA orthologs (16). Because mitochondria are likely to contribute to the hypoxia͞reoxygenation-mediated increase in ROS (6,44,45), preferential localization of at least some MSRA in or near mitochondria is consistent with this enzyme functioning to repair any oxidative damage in this organelle.…”

The different apoptotic potential of the p53 codon 72 alleles increases with age and modulates in vivo ischaemia-induced cell death

“…We found that Arg þ subjects have higher serum levels of cardiac Troponin I and CK-MB, two qualitative/quantitative markers for the extent of the ischaemic damage at the myocardial level. 38 This finding can be better understood by taking into account that hypoxia-induced cell death mediates an oxidative stressinduced damage at the mitochondrial level in cardiomyocytes, 42 and it induces the targeting of p53 to the mitochondrion. 2 Moreover, it is known that p53 activates mitochondrialdependent death pathway in ventricular myocytes.…”

Prohibitin protects against hypoxia-induced H9c2 cardiomyocyte cell death

“…Significant and irreversible decrease in the mitochondrial membrane potential during ischemia and oxidative stress in cardiomyocytes was reported (15). To investigate the mechanism of rescue effect by prohibitin, we analyzed mitochondrial membrane potential levels (A) H9c2 cells were fractionated using differential centrifugation method.…”

“…Although the reason for the difference is unclear, it may be derived from the difference of experimental conditions, hypoxia or H 2 O 2 . Mitochondrial electron transport chain increases in generation of reactive oxygen species in response to hypoxia despite the conditions of low O 2 concentration (11,15). It is proposed that hypoxia induces increase in superoxide generation in the mitochondrial matrix, which can be converted to H 2 O 2 by Mn-superoxide dismutase (5).…”