Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes.
Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 +/- 3% to 14 +/- 2%. Return of contraction was observed in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H2O2) increased approximately 2.5-fold. As a substitute for hypoxia, the addition of H2O2 (15 micromol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic acid, and the anion channel inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, all of which also abrogated protection. ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor L-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection. Furthermore, mitochondrial anion channels and cytosolic dismutation to H2O2 may be important steps for oxidant induction of hypoxic preconditioning.
Vanden Hoek
T L
TL
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Becker
L B
LB
Shao
Z
Z
Li
C
C
Schumacker
P T
PT
D000818:Animals; D016923:Cell Death; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002642:Chick Embryo; D003600:Cytosol / Q000201:enzymology; D004050:Ditiocarb / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019157:Ischemic Preconditioning, Myocardial; D008689:Methacrylates; D008929:Mitochondria, Heart / Q000378:metabolism*; D009200:Myocardial Contraction; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009861:Onium Compounds / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D008625:Tiopronin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008689:Methacrylates; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D013844:Thiazoles; C007517:diphenyleneiodonium; C030517:myxothiazol; D004050:Ditiocarb; D006861:Hydrogen Peroxide; D008625:Tiopronin; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase