Several hypotheses on the mechanisms of ischemic preconditioning involve activation of channels or transporter proteins affecting the ion flux, most notably for K+ and H+, across the inner mitochondrial membrane. Increasing mitochondrial K+ or H+ conductance will have several physiological consequences. The aim of the present work was to explore how some of these alterations relate to commonly proposed mechanisms for ischemia-reperfusion injury in heart mitochondria.
An extensive uncoupling will cause a dramatic effect on normal mitochondrial function, but so-called mild uncoupling has been suggested to reduce the sensitivity of mitochondria to undergo calcium-induced mPT by limiting the calcium uptake and ROS production of mitochondria [12, 25]. The CRC assay is a sensitive and quantitative method for evaluating mitochondrial susceptibility to mPT induction , and by simultaneous monitoring of respiration the specificity is increased . Here, we demonstrate that heart mitochondrial CRC is dose-dependently reduced by uncoupling irrespective of whether H+ or K+ conductance is increased, and that the reduced CRC is caused by mPT (as demonstrated by the respiratory decrease). There was no tendency of any beneficial effect at lower concentrations of the K+ carrier valinomycin or the protonophore CCCP. At the highest tested concentration of CCCP, 200 nM, the respiratory increase was less than a state 2 to state 3 transition but the ability to retain Ca2+ was virtually abolished and mitochondria underwent permeability transition at a very low calcium load. Ca2+ is the principal trigger of mPT and a reduction in matrix Ca2+ would arguably reduce mPT activation. However, it is the free Ca2+ ions that trigger mPT and most calcium in mitochondria is retained as inactive calcium-phosphate complexes . The formation of calcium-phosphates is highly dependent on an alkaline matrix environment. A small decrease in ∆pH, as occurs with uncoupling following H+ entry, will increase the concentration of free Ca2+ even if the electrophoretic driving force for Ca2+ uptake is reduced. Uncoupling may also directly increase the propensity of the mPT pore to open due to a more oxidized redox state and a reduced mitochondrial membrane potential (∆Ψm) [27, 28].
Increasing K+ flux into the mitochondria may affect several factors important for calcium handling and mPT regulation, including ∆Ψm, ∆pH, ROS generation and matrix volume, which are somewhat different to increasing H+ flux. For example, increasing K+ or H+ conductance may be expected to have opposite effects on ∆pH due to activation of the K+/H+ exchanger by the former. Whether an alteration in any of these parameters is beneficial or not to the resistance of mitochondria to the dramatic consequences of calcium overload depend both on their direct and potential indirect effects on the mPT pore components, as pointed out above for the effect of ∆pH on free Ca2+. In a previous study, we found that low increases in K+ but not H+ conductance was beneficial to brain mitochondrial resistance to calcium-induced permeability transition, and that the beneficial effect probably was mediated by an increase in matrix volume . In the present study, there was no qualitative difference between increasing H+ or K+ conductance on CRC. The negative effects of uncoupling thus predominate over other potential direct beneficial effects of increasing K+ conductance in rat heart mitochondria.
Changes in mitochondrial matrix volume influence respiratory function [29–31] and we have previously demonstrated that increased matrix volume in brain mitochondria either caused by an elevated influx of K+ or by suspending mitochondria in hypoosmolar KCl medium increases CRC . In the present study we explored respiratory changes and CRC in heart mitochondria suspended in hypo- or hyperosmolar KCl media. Increasing medium osmolarity by KCl (or LiCl) dose-dependently decreased CRC indicating a positive correlation between matrix volume and CRC. A possible confounder to increasing osmolarity with KCl or LiCl is the simultaneous change in ionic strength which may affect e.g. cytochrome c charge interactions . However, increasing medium osmolarity with sucrose likewise decreased CRC. In contrast to the effects of increasing medium osmolarity, decreasing the osmolarity further than the standard conditions did not cause the expected increase in CRC. A plausible explanation may be the reduced respiratory capacity of heart mitochondria in the hypoosmolar medium. An increase in matrix volume has been shown to cause release of cytochrome c thus reducing respiratory capacity in isolated liver mitochondria . The same authors also found a reduced calcium uptake in mitochondria following exposure to a hypoosmotic shock. In a limited set of experiments in heart mitochondria (data not shown) we found that there was a tendency for exogenously added cytochrome c to increase respiration more in hypoosmolar media compared to hyperosmolar conditions, but these findings were not explored further. It is thus plausible that any beneficial effect on heart mitochondrial CRC by an enlarged matrix volume following increased K+ flux is counteracted by a respiratory inhibition and a direct negative effect of uncoupling. Even though the majority of studied characteristics described herein were similar to previous findings in brain mitochondria, there seems to be a distinct organ specific difference in relation to the response to increased K+ conductance and matrix volume.
ROS production of mitochondria has since long been recognized to increase with elevated proton-motive force [34, 35]. This is particularly evident when isolated mitochondria are supplemented with solely succinate as respiratory substrate to support so-called reverse electron transport through respiratory complex I . Ischemic preconditioning reduces oxidative damage in mitochondria, and a reduction of oxidative stress may mediate a reduced propensity for mPT activation . Previous studies have demonstrated both increased and decreased ROS generation following augmented K+ flux in mitochondria [15–19]. In the present study, both increased K+ and H+ flux dose-dependently reduced the production of H2O2 in heart mitochondria using complex I substrates. There was no apparent qualitative or quantitative difference between increased K+ and H+ flux; rather, the level of ROS reduction was in proportion to the extent of uncoupling. However, the reduced ROS generation did not translate into an increased resistance to mPT induction.
The capacity of mitochondria to detoxify ROS is less explored but likely equally or more important than mitochondrial ROS production in oxidative stress  and there is no clear evidence whether mitochondria function as net source or sink of ROS . Heart mitochondria are equipped with effective antioxidant systems for scavenging of ROS and dysfunction of these are involved in cardiovascular disease . Malfunction of mitochondrial thioredoxin reductase causes dilated cardiomyopathy in man  and aggravates ischemia-reperfusion injury in mice , while overexpression of glutaredoxin-2 reduces ischemia-reperfusion injury in mice . The mitochondrial antioxidative systems are dependent on intact respiratory function. Reduced glutathione and NADPH provide antioxidant defences against H2O2 mediated damage through the action of e.g. the glutathione peroxidase family, peroxiredoxins and glutaredoxins. The reduction of GSH and thioredoxin reductase is dependent on NADPH generation from e.g. the proton motive force-driven transhydrogenase . Although uncoupling of mitochondrial respiration reduces ROS production, any interference with ROS scavenging may result in an overall negative effect on mitochondrial ROS handling. Therefore, we investigated the heart mitochondrial capacity of H2O2 removal under conditions of uncoupling and calcium overload. The mitochondrial H2O2 scavenging rate was more than 10-fold greater than the detected production rate of H2O2 under standard conditions. Whereas uncoupling decreased H2O2 production it did not affect H2O2 scavenging. In contrast, calcium overload leading to mPT dramatically reduced H2O2 scavenging in line with previous reports [23, 24]. Any effects of calcium unrelated to mPT activation were not explored in the present study.
We did not detect any specific effect of the MitoKATP opener diazoxide. The compound uncoupled respiration at high concentrations, which also has been demonstrated previously . The present experiments were not primarily designed to explore the putative MitoKATP. Such experiments are usually performed within the first few minutes upon incubation in experimental medium and the present experiments examining general effects of increased H+ and K+ conductance required a longer time frame. It has been noted that the MitoKATP is sensitive to rapid inactivation  and there is also a current debate over its existence .