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

Adenosine prevents TNFα-induced decrease in endothelial mitochondrial mass via activation of eNOS-PGC-1α regulatory axis.

We tested whether adenosine, a cytoprotective mediator and trigger of preconditioning, could protect endothelial cells from inflammation-induced deficits in mitochondrial biogenesis and function. We examined this question using human microvascular endothelial cells exposed to TNFα. TNFα produced time and dose-dependent decreases in mitochondrial membrane potential, cellular ATP levels, and mitochondrial mass, preceding an increase in apoptosis. These effects were prevented by co-incubation with adenosine, a nitric oxide (NO) donor, a guanylate cyclase (GC) activator, or a cell-permeant cyclic GMP (cGMP) analog. The effects of adenosine were blocked by a nitric oxide synthase inhibitor, a soluble guanylate cyclase inhibitor, a morpholino antisense oligonucleotide to endothelial nitric oxide synthase (eNOS), or siRNA knockdown of the transcriptional coactivator, PGC-1α. Incubation with exogenous NO, a GC activator, or a cGMP analog reversed the effect of eNOS knockdown, while the effect of NO was blocked by inhibition of GC. The protective effects of NO and cGMP analog were prevented by siRNA to PGC-1α. TNFα also decreased expression of eNOS, cellular NO levels, and PGC-1α expression, which were reversed by adenosine. Exogenous NO, but not adenosine, rescued expression of PGC-1α in cells in which eNOS expression was knocked down by eNOS antisense treatment. Thus, TNFα elicits decreases in endothelial mitochondrial function and mass, and an increase in apoptosis. These effects were reversed by adenosine, an effect mediated by eNOS-synthesized NO, acting via soluble guanylate cyclase/cGMP to activate a mitochondrial biogenesis regulatory program under the control of PGC-1α. These results support the existence of an adenosine-triggered, mito-and cytoprotective mechanism dependent upon an eNOS-PGC-1α regulatory pathway, which acts to preserve endothelial mitochondrial function and mass during inflammatory challenge

Effects of TNFα on markers of mitochondrial mass and expression of several effector proteins for mitochondrial biogenesis.

<p>In separate experiments, HMEC-1 cells in 100 cm plates were incubated with TNFα (1 ng/ml), then 4 separate indices of mitochondrial content were examined. (<b>A</b>) Time course of effect of TNFα on mitochondrial mass as measured using a fluorescent plate assay for uptake of MTG dye. At the conclusion of TNFα incubation period, cells were loaded with MTG as described in Methods, then harvested and equal aliquots were separately analyzed for MTG fluorescence and total protein. Mitochondrial mass was expressed as MTG fluorescence, normalized for protein content. Experiments were repeated 3–4 times per time point. * denotes significant decrease in apparent mass compared with respective time point control (P<0.0001). (<b>B</b>) Cells prepared as described for panel (A) were incubated in the presence or absence of TNFα (1 ng/ml) for 48 h. Cells were harvested and total DNA was isolated, purified, and subjected to quantitative PCR as described in Methods, using primer sets for both nuclear and mitochondrial DNA (nDNA and mtDNA, respectively). Values are expressed as the mtDNA/nDNA ratio, for 5 separate replications of the experiment. * denotes significant difference from control, p<0.01). (<b>C</b>) Cells prepared and treated as described for panel (B) were harvested and lysates were analyzed for citrate synthase activity as described in Methods. Values summarize the results of 4 separate experiments, * denotes significant difference from control, p<0.05. (<b>D</b>) Cells prepared and treated as described for panel (B) were harvested and lysates were subjected to SDS-PAGE of equal amounts of lysate protein, followed by western blot anlaysis of the indicated proteins. Left-hand panel shows representative blot from among 3 separate experiments, right-hand panel shows semi-quantitative analysis of band density from the full dataset from all experiments. Except for GAPDH, TNFα elicited significantly decreased expression of all proteins examined, p values for each are shown in the figure.</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

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