Document Type

Article

Language

eng

Publication Date

3-2013

Publisher

Elsevier

Source Publication

Free Radical Biology and Medicine

Source ISSN

0891-5849

Original Item ID

DOI: 10.1016/j.freeradbiomed.2012.09.020

Abstract

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca2+ uptake. Here we sought to elucidate the effects of extramitochondrial Ca2+ (e[Ca2+]) on ROS production (measured as H2O2 release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl2 and then energized with the complex I substrate Na+ pyruvate or the complex II substrate Na+ succinate. Mitochondrial H2O2 release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca2+] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H2O2 (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca2+], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H2O2 release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca2+] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H2O2 emission from complex III during ischemia.

Comments

NOTICE: this is the author’s version of a work that was accepted for publication in Free Radical Biology and Medicine. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Free Radical Biology and Medicine, Vol. 56 (March 2013): 193–203. DOI. © 2013 Elsevier. Used with permission.

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