Hypothermia augments reactive oxygen species detected in the guinea pig isolated perfused heart

Hypothermic perfusion of the heart decreases oxidative phosphorylation and increases NADH. Because O-2 and substrates remain available and respiration ( electron transport system, ETS) may become impaired, we examined whether reactive oxygen species (ROS) exist in excess during hypothermic perfusion. A fiberoptic probe was placed on the left ventricular free wall of isolated guinea pig hearts to record intracellular ROS, principally superoxide (O-2(-.)), and an extracellular reactive nitrogen reactant, principally peroxynitrite (ONOO-), a product of nitric oxide (NO.) + O-2(-.). Hearts were loaded with dihydroethidium (DHE), which is oxidized by O-2(-.) to ethidium, or were perfused with L-tyrosine, which is oxidized by ONOO- to dityrosine (diTyr). Shifts in fluorescence were measured online; diTyr fluorescence was also measured in the coronary effluent. To validate our methods and to examine the source and identity of ROS during cold perfusion, we examined the effects of a superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid) porphyrin chloride ( MnTBAP), the nitric oxide synthase inhibitor N-G-nitro-L-arginine methyl ester (L-NAME), and several agents that impair electron flux through the ETS: menadione, sodium azide (NaN3), and 2,3-butanedione monoxime (BDM). Drugs were given before or during cold perfusion. ROS measured by DHE was inversely proportional to the temperature between 37 degreesC and 3 degreesC. We found that perfusion at 17 degreesC increased DHE threefold versus perfusion at 37 degreesC; this was reversed by MnTBAP, but not by L-NAME or BDM, and was markedly augmented by menadione and NaN3. Perfusion at 17 degreesC also increased myocardial and effluent diTyr ( ONOO-) by twofold. L-NAME, MnTBAP, or BDM perfused at 37 degreesC before cooling or during 17 degreesC perfusion abrogated, whereas menadione and NaN3 again enhanced the cold-induced increase in ROS. Our results suggest that hypothermia moderately enhances O-2(-.) generation by mitochondria, whereas O-2(-.) dismutation is markedly slowed. Also, the increase in O-2(-.) during hypothermia reacts with available NO. to produce ONOO-, and drug-induced O-2(-.) dismutation eliminates the hypothermia-induced increase in O-2(-.).