Abstract
BackgroundIschemic preconditioning has been proposed to involve changes in mitochondrial H+ and K+ fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K+ channels (MitoKATP). The objectives of the present study were to explore how increased H+ and K+ fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT).ResultsIsolated rat heart mitochondria were exposed to a wide concentration range of the protonophore CCCP or the potassium ionophore valinomycin to induce increased H+ and K+ conductance, respectively. Simultaneous monitoring of mitochondrial respiration and calcium retention capacity (CRC) demonstrated that the relative increase in respiration caused by valinomycin or CCCP correlated with a decrease in CRC, and that no level of respiratory uncoupling was associated with enhanced resistance to mPT. Mitochondria suspended in hyperosmolar buffer demonstrated a dose-dependent reduction in CRC with increasing osmolarity. However, mitochondria in hypoosmolar buffer to increase matrix volume did not display increased CRC. ROS generation was reduced by both K+- and H+-mediated respiratory uncoupling. The ability of heart mitochondria to detoxify H2O2 was substantially greater than the production rate. The H2O2 detoxification was dependent on respiratory substrates and was dramatically decreased following calcium-induced mPT, but was unaffected by uncoupling via increased K+ and H+ conductance.ConclusionIt is concluded that respiratory uncoupling is not directly beneficial to rat heart mitochondrial resistance to calcium overload irrespective of whether H+ or K+ conductance is increased. The negative effects of respiratory uncoupling thus probably outweigh the reduction in ROS generation and a potential positive effect by increased matrix volume, resulting in a net sensitization of heart mitochondria to mPT activation.
Highlights
Ischemic preconditioning has been proposed to involve changes in mitochondrial H+ and K+ fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K+ channels (MitoKATP)
We explored the relation between heart mitochondrial matrix volume and the mitochondrial resistance to calcium-induced mitochondrial permeability transition (mPT) by infusing calcium to mitochondria suspended in media with different osmolarities
Increasing mitochondrial matrix volume by reducing the KCl concentration and osmolarity of the media from 125 mM to 75 mM had no apparent effect on calcium retention capacity (CRC) but state 3 respiration and the corresponding respiratory control ratio (RCR) were reduced
Summary
Ischemic preconditioning has been proposed to involve changes in mitochondrial H+ and K+ fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K+ channels (MitoKATP). The objectives of the present study were to explore how increased H+ and K+ fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT). Several different pathways involving altered H+ or K+ entry into the mitochondrial matrix have been proposed to modify resistance to ischemia-reperfusion injury. -called mild uncoupling [11] has been suggested to reduce the sensitivity of mitochondria to undergo calciuminduced mPT both by reducing the ΔΨm-dependent calcium uptake and by attenuating reactive oxygen species (ROS) production of mitochondria [12,13]. We have previously demonstrated that enhanced matrix volume in brain mitochondria increased their resistance to calcium-induced mPT [21]
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