Altered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQ(®) and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased expression of UCP-2, Complex IV (cytochrome c oxidase) and Complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (ΔG(0)), were similar at baseline but had decreased by the end of low-flow ischemia (-57.0 ± 2.1 versus -51.1 ± 1.4 kJ/mol; P < 0.05). In conclusion, within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation.

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Myocardial uncoupling protein (UCP)-2 is increased with chronic peroxisome proliferator-activated receptor γ (PPARγ) stimulation but the effect on membrane potential and superoxide is unclear. Wild type (WT) and UCP-2 knock-out (KO) mice were given a 3-week diet of control (C) or the PPAR γ agonist pioglitazone (50 μg/gram-chow per day) (PIO). In isolated mitochondria, UCP-2 content by Western blots, membrane potential (ΔΨm) by tetraphenylphosphonium (TPP) and relative superoxide levels by dihydroethidium (DHE) were measured. Oxygen respiration was determined at baseline and following 10 minutes anoxia-reoxygenation. PIO induced a 2-fold increase in UCP-2 and nuclear-bound PGC1α in WT mice with no UCP-2 expression in KO mice. Mitochondrial ΔΨm from WT mice on C and PIO diets was −166±4 mv and −147±6 mV respectively (P<0.05) and were lower than UCP-2 KO mice on C and PIO (−180±4 and −180±4 mv respectively; P<0.05). Maximal complex III inhibitable superoxide from WT mice on C and PIO diets was 22.5±1.3 and 17.8±1.1 AU respectively (P<0.05) and were lower than UCP-2 KO on C and PIO (32.9±2.3 and 29.2±1.9 AU respectively; P<0.05). Post-anoxia, the respiratory control index (RCI) in mitochondria from WT mice with and without PIO was 2.5±0.3 and 2.4±0.2 respectively and exceeded that of UCP-2 KO mice on C and PIO (1.2±0.1 and 1.4±0.1 respectively (P<0.05). In summary, chronic PPARγ stimulation leads to depolarization of the inner membrane and reduced superoxide of isolated heart mitochondria, which was critically dependent upon increased expression of UCP-2. UCP-2 expression affords resistance to brief anoxia-reoxygenation.

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