. 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...

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.

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Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O 2 that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H 2 O 2 generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.Previous studies have examined the cellular consequences of tissue ischemia, but the molecular mechanisms that regulate cell death in this syndrome are still not fully understood. When blood flow to a tissue is interrupted, the extent of injury is influenced by the severity and duration of the ischemic challenge such that longer periods of ischemia are associated with more extensive cell death (1, 2). Restoration of blood flow relieves the ischemic stress, although morphological evidence of tissue injury appears only after the oxygen supply has been restored (3). This has led to the widely accepted notion that reperfusion is responsible for the cellular damage in ischemia-reperfusion injury (4). However, the events during ischemia that prime the cell for death during reperfusion are not fully understood.Oxidant stress arising from excessive production of reactive oxygen species (ROS) 3 has long been associated with ischemia-reperfusion injury (4, 5). In this regard, animals with genetic impairment of antioxidant systems exhibit increased susceptibility to myocardial ischemia-reperfusion injury (6); antioxidants administered during ischemia-reperfusion confer protection against subsequent cell death (7-9); and overexpression of antioxidant enzymes confers protection against ischemia-reperfusion injury (10, 11). Multiple studies have demonstrated that a burst of oxidant stress is generated upon reperfusion of ischemic cardiomyocytes (5, 12, 13). Int...

The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the mutations in a high-throughput manner. We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous mutations using the mixed-all-nominated-in-one (MANO) method. G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent mutation in lung cancer. We surveyed the prevalence of concurrent mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib. Several mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers.We identified several mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. .

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We used a mobile computed tomography (CT) unit for postmortem examinations of deceased subjects to see how many mistakes on cause-of-death diagnoses were made in Japan. In 5 of 20 cases, the cause of death determined by CT was different from the diagnosis made by superficial postmortem examination. In one case, the superficial examination suggested no trauma, whereas a subdural hematoma was found on cranial CT images. We concluded that postmortem examinations in Japan were not effective when screening for crimes or accidents. Using a mobile CT scanner in postmortem examination may be a viable method of screening for causes of deaths, although it cannot be used as a substitute for autopsy.