We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca(2+)-sensitive K(+) (mtK(Ca)) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK(Ca) channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K(+) influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (DeltaPsi(m)). To test this hypothesis, we measured effects of NS-1619, a putative mtK(Ca) channel opener, and valinomycin, a K(+) ionophore, on mitochondrial respiration, DeltaPsi(m), and ROS generation in guinea pig heart mitochondria. NS-1619 (30 microM) increased state 2 and 4 respiration by 5.2 +/- 0.9 and 7.3 +/- 0.9 nmol O(2).min(-1).mg protein(-1), respectively, with the NADH-linked substrate pyruvate and by 7.5 +/- 1.4 and 11.6 +/- 2.9 nmol O(2).min(-1).mg protein(-1), respectively, with the FADH(2)-linked substrate succinate (+ rotenone); these effects were abolished by the mtK(Ca) channel blocker paxilline. DeltaPsi(m) was not decreased by 10-30 microM NS-1619 with either substrate, but H(2)O(2) release was increased by 44.8% (65.9 +/- 2.7% by 30 muM NS-1619 vs. 21.1 +/- 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H(2)O(2) release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained DeltaPsi(m), despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K(+) influx during states 2 and 4 increases mitochondrial respiration while maintaining DeltaPsi(m); this allows singlet electron uptake by O(2) and ROS generation.

NADH increases during ischemia because O2 shortage limits NADH oxidation at the electron transport chain. Ischemic (IPC) and anesthetic preconditioning (APC) attenuate cardiac reperfusion injury. We examined whether IPC and APC similarly alter NADH, i.e., mitochondrial metabolism. NADH fluorescence was measured at the left ventricular wall of 40 Langendorff-prepared guinea pig hearts. IPC was achieved by two 5-min periods of ischemia and APC by exposure to 0.5 or 1.3 mM sevoflurane for 15 min, each ending 30 min before 30 min of global ischemia. During ischemia, NADH initially increased in nonpreconditioned control hearts and then gradually declined below baseline levels. This increase in NADH was lower after APC but not after IPC. The subsequent decline was slower after IPC and APC. On reperfusion, NADH was less decreased after IPC or APC, mechanical and metabolic functions were improved, and infarct size was lower compared with controls. Our results indicate that IPC and APC cause distinctive changes in mitochondrial metabolism during ischemia and thus lead to improved function and tissue viability on reperfusion.experimental; infarction; sevoflurane; dose dependency; mitochondrial function EXPERIMENTS IN ISOLATED MYOCYTES, intact hearts, and whole animal models have shown that cardiac cell injury and mechanical dysfunction are caused by reperfusion after prolonged ischemia. Depending on the duration and magnitude of the ischemia, the injury can be reversible (stunning) or irreversible (infarction) (20). Several interrelated mechanisms are responsible:

Reactive oxygen species (ROS) are implicated in triggering anesthetic preconditioning (APC). The ROS superoxide (O(2)(.-)) was measured continuously in guinea pig isolated hearts. Sevoflurane directly increased O(2)(.-) but led to attenuated O(2)(.-) formation during ischemia. This demonstrates triggering of APC by ROS and clarifies the mechanism of cardioprotection during ischemia.

Heinen A, Aldakkak M, Stowe DF, Rhodes SS, Riess ML, Varadarajan SG, Camara AK. Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca 2ϩ -sensitive K ϩ channels. Am J Physiol Heart Circ Physiol 293: H1400-H1407, 2007. First published May 18, 2007; doi:10.1152/ajpheart.00198.2007.-Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O2 concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH2-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O2•Ϫ ); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate ϩ rotenone-dependent H 2O2 production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca 2ϩ -sensitive K ϩ channel (mtBKCa) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O 2 consumption, membrane potential (⌬⌿ m), H2O2 release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 M) increased state 2 and state 4 respiration by 26 Ϯ 4% and 14 Ϯ 4%, respectively; this increase was abolished by the BK Ca channel blocker paxilline (5 M). Paxilline alone had no effect on respiration. NS-1619 did not alter ⌬⌿ m or redox state but decreased H 2O2 production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K ϩ influx through mtBKCa channels reduces mitochondrial H2O2 production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H 2O2 production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K ϩ influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.

In this study, we determined 1) whether ROS scavenging only during cold perfusion before global ischemia improves mitochondrial and myocardial function, and 2) which ROS leads to compromised cardiac function during ischemia and reperfusion (I/R) injury. Using fluorescence spectrophotometry, we monitored redox balance (NADH and FAD), O 2•Ϫ levels and mitochondrial Ca 2ϩ (m[Ca 2ϩ ]) at the left ventricular wall in 120 guinea pig isolated hearts divided into control (Con), MnTBAP (a superoxide dismutase 2 mimetic), MnTBAP (M) ϩ catalase (C) ϩ glutathione (G) (MCG), CϩG (CG), and N G -nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase inhibitor) groups. After an initial period of warm perfusion, hearts were treated with drugs before and after at 27°C. Drugs were washed out before 2 h at 27°C ischemia and 2 h at 37°C reperfusion. We found that on reperfusion the MnTBAP group had the worst functional recovery and largest infarction with the highest m [Ca 2ϩ ], most oxidized redox state and increased ROS levels. The MCG group had the best recovery, the smallest infarction, the lowest ROS level, the lowest m [Ca 2ϩ ], and the most reduced redox state. CG and L-NAME groups gave results intermediate to those of the MnTBAP and MCG groups. Our results indicate that the scavenging of cold-induced O 2•Ϫ species to less toxic downstream products additionally protects during and after cold I/R by preserving mitochondrial function. Because MnTBAP treatment showed the worst functional return along with poor preservation of mitochondrial bioenergetics, accumulation of H 2 O 2 and/or hydroxyl radicals during cold perfusion may be involved in compromised function during subsequent cold I/R injury. hypothermic ischemia; mitochondrial Ca 2ϩ ; reactive oxygen species HYPOTHERMIA of the arrested, ischemic heart improves its function on reperfusion compared with the normothermic arrested ischemic heart. The strategy behind hypothermic protection against ischemia, i.e., better tissue perfusion, and improved metabolic and mechanical function on reperfusion, is the reduced mitochondrial respiration and oxidative phosphorylation during ischemia that results in better mitochondrial respiration and regeneration of ATP on reperfusion. For example, we demonstrated that NADH, m [Ca 2ϩ ], and reactive O 2 species (ROS) levels were less altered during and after 30 min of ischemia at 17°C vs. 37°C (39). We reported that the more severe the hypothermia, the later is the onset of deleterious changes in mitochondrial function (1,2,16,22,31).Although hypothermia is very protective against ischemia, hypothermic perfusion, e.g., before subsequent cardiac ischemia, may cause injury due to altered cellular ion homeostasis resulting from impaired membrane ion pumps and exchangers and/or to reduced activity of enzymes responsible for mitochondrial respiration, scavenging of ROS, and contractile activity. A well-known effect of hypothermia is hypercontracture with elevated cytosolic [Ca 2ϩ ] (42). Another is a decreasing temperature-depende...