Mitochondrial reactive oxygen species: A double edged sword in ischemia/reperfusion vs preconditioning

Reductions in the blood supply produce considerable injury if the duration of ischemia is prolonged. Paradoxically, restoration of perfusion to ischemic organs can exacerbate tissue damage and extend the size of an evolving infarct. Being highly metabolic organs, the heart and brain are particularly vulnerable to the deleterious effects of ischemia/reperfusion (I/R). While the pathogenetic mechanisms contributing to I/R-induced tissue injury and infarction are multifactorial, the relative importance of each contributing factor remains unclear. However, an emerging body of evidence indicates that the generation of reactive oxygen species (ROS) by mitochondria plays a critical role in damaging cellular components and initiating cell death. In this review, we summarize our current understanding of the mechanisms whereby mitochondrial ROS generation occurs in I/R and contributes to myocardial infarction and stroke. In addition, mitochondrial ROS have been shown to participate in preconditioning by several pharmacologic agents that target potassium channels (e.g., ATP-sensitive potassium (mKATP) channels or large conductance, calcium-activated potassium (mBKCa) channels) to activate cell survival programs that render tissues and organs more resistant to the deleterious effects of I/R. Finally, we review novel therapeutic approaches that selectively target mROS production to reduce postischemic tissue injury, which may prove efficacious in limiting myocardial dysfunction and infarction and abrogating neurocognitive deficits and neuronal cell death in stroke

Time and TNFα dose-dependent changes in HMEC-1 apoptosis and mitochondrial membrane potential (Ψ), and modulation of TNFα effects by adenosine.

<p>(<b>A</b>) HMEC-1 cells on gelatin-coated glass cover slips were incubated with TNFα at 1 or 10 ng/ml for the indicated period. At each time point, apoptotic cells were fixed, mounted, and stained with DAPI, then counted as described in Methods; apoptosis calculated as a percentage of total cells in 6 fields of view, with 200 cells counted per 40X field. Data analyzed by two-way ANOVA with multiple comparisons using a general linear model. Experiments were repeated 3–4 times per treatment group. At a given time of incubation, * indicates significantly different from respective time point control, P<0.01. (<b>B</b>) HMEC-1 cells in 24-well plates were incubated with TNFα for the indicated times, loaded with TMRM or MTG dyes, then harvested for measurement of Ψ or total mitochondrial mass, respectively, as described in Methods. Data are means ± SEM for 8 replicates for each treatment/time combination, repeated 4 separate times. Data were analyzed as described for panel (A). All TNFα values were significantly different from their respective controls at each time point, differing letters denote significant TNFα dose effects, P<0.05. (<b>C, D</b>) Attenuation of TNFα effects on apoptosis and Ψ, respectively, by adenosine. Cells set up as described for panels (A) & (B) were incubated with or without TNFα (1 or 10 ng/ml), with or without co-incubation with adenosine (Ado, 10 uM) for either 48 or 72 h, then apoptosis or Ψ were measured. Results are expressed as the % change from the respective time point controls. Data are means ± SEM for 4 separate repititions of each experiment. At both time points, all TNFα values were significantly different from respective control values (P<0.001), apoptosis values in response to Ado+TNFα were significantly higher than control at 48 h (TNFα, 10 ng/ml) and at 72 h (both doses of TNFα), p<0.05. * denote significant attenuation of TNFα-induced effect at each time point in response to Ado (P<0.001).</p

Effect of adenosine/NO is mediated by a sGC/cGMP-dependent mechanism.

<p>Both sets of experiments (A, B) were repeated 3 times. (<b>A</b>) Cells in 100 cm dishes were incubated for 48 h with TNFα±Ado, in the presence or absence of sGC inhibitor, ODQ (30 µM), sGC agonist, YC-1 (100 µM) or cGMP analog, 8-Br-cGMP (500 µM). Mitochondrial mass measured using MTG fluorescence. Differing letters denote significant between-group differences, p<0.01. (<b>B</b>) Cells (non-transfected, or transfected with NOS3 or SON3 morpholino oligos to eNOS) were incubated for 48 h with TNFα±detaNO (100 nM) in the presence or absence of ODQ, YC-1, or 8-Br-cGMP, then MTG fluorescence was measured. Differeing letters denote significant between-group differences, p<0.05.</p

Effect of adenosine (Ado) is mediated by eNOS/NO-dependent mechanism.

<p>(<b>A</b>) Cells were prepared and treated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098459#pone-0098459-g003" target="_blank">Figure 3</a>, except that in one group, cells were preincubated for 15 min with L-NIO (100 µM) prior to addition of Ado and TNFα. Mitochondrial mass was assayed using MTG fluorescence as described for previous figures. Values reported are from 3 separate replications of the experiment per group, differing letters denote significant between-group differences, P<0.05. (<b>B</b>) Western blot of total eNOS expression in response to TNFα vs. Ado+TNFα; blot shown is from the same experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098459#pone-0098459-g003" target="_blank">Figure 3B</a>. (<b>C</b>) Cells were incubated with TNFα in the presence or absence of graded concentrations of the NO donor, detaNO, followed by measurement of MTG fluorescence. Differing letters denote significant dose-dependent differences (p<0.05). Experiment was repeated 4 times. (<b>D</b>) Upper panel: western blot of HMEC-1 total eNOS expression, 48 h after transfection with either morpholino eNOS antisense (NOS3) or invert control (SON3) oligonucleotides. Lower panel: MTG fluorescence in cells treated with TNFα±Ado in either non-transfected cells or cells transfected with control or eNOS antisense morpholino oligos. Experiment was repeated 4 times per group, differing letters denote significant between-group differences (p<0.01).</p

Effects of adenosine/NO are mediated by a PGC-1α-dependent mechanisim.

<p>(<b>A</b>) Western blot of expression of PGC-1α, Nrf-2, eNOS, and GAPDH, 48 h after transfection with either control or PGC-1α-specific siRNA. (<b>B</b>) Western blot of PGC-1α expression in response to TNFα±Ado or detaNO in either non-transfected cells, or cells transfected with either control (SON3) or eNOS antisense (NOS3) morpholino oligonucleotides. Blot shown is representative of 3 separate experiments. (<b>C</b>) MTG fluorescence after 48 h incubation with TNFα±Ado, detaNO, or 8-Br-cGMP in either control or PGC-1α siRNA-transfected cells (PGC siRNA). Data are from 4 separate experiments for reach group, differing letters denote significant between-group differences, p<0.05. (<b>D</b>) Measurement of Ψ in HMEC-1 cells in 24-well plates, treated as indicated, then loaded with TMRM or MTG dyes, as described in Methods. Data are means ± SEM for 4 replicates for each treatment/time combination, repeated 3 separate times. Asterisks denote values significantly different from control value, *: P<0.05, **: p<0.01.</p

Control studies of effect of individual treatments (48 h) on MTG fluorescence.

<p>Indicated treatments were as described for other figures. Values are means ± SEM of 4 separate experiments per treatment except for NOS3/SON3 where n = 5 and PGC-1α/Control siRNA where n = 3. Differing letters denote significant between-group differences.</p