Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion

Am J Physiol Heart Circ Physiol. 2014 Oct 1;307(7):H996-H1004. doi: 10.1152/ajpheart.00189.2014. Epub 2014 Aug 1.


Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H(+) leak decreases ROS formation; it has been postulated that increasing H(+) leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H(+) leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H(+) leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H(+) leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H(+) leak. H(+) leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H(+) leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H(+) leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Keywords: ischemia-reperfusion; mitochondria; proton leak; reactive oxygen species; uncoupling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / pharmacology
  • Male
  • Membrane Potential, Mitochondrial
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Myocardial Reperfusion Injury / metabolism*
  • Protons*
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism*
  • Uncoupling Agents / pharmacology*


  • Protons
  • Reactive Oxygen Species
  • Uncoupling Agents
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone