Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning

Abstract

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.

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection

“…This model was previously reported and a portion of the data in the present report has been reproduced with permission [11]. This includes the regional blood flow measurements, high-energy nucleotides from biopsy samples and respiration from isolated mitochondrial samples.…”

“…These studies suggest that both hibernation and ischemic preconditioning have evolved adaptations that result in preserved energetics during oxygen deprivation. The expression of uncoupling protein-2 for instance, is increased in isolated mitochondria from hibernating hearts [14] as well as ischemic preconditioned hearts during SWOP [11,22], and may be an adaptation to reduce excess accumulation of reactive oxygen species during repetitive supply-demand ischemia.…”

“…Mitochondria were isolated from fresh cardiac tissue as described in detail previously [11,14]. In brief, tissue (~5 g samples) was extracted from the LAD region of SWOP and HIB pigs and immediately placed in ice-cold mitochondrial isolation buffer (MIB), pH 7.15, containing 50 mM sucrose, 200 mM mannitol, 1 mM EGTA, 5 mM KH 2 PO 2 , 5 mM MOPS and 0.1 % fatty acid free BSA.…”

“…The free energy of ATP hydrolysis was determined from transmural biopsies with analysis of high-energy nucleotides and was more favorable in SWOP compared with SHAM pigs. A portion of these data has been previously published [11] and is reproduced with permission.…”

Expression of uncoupling protein-2 remains increased within hibernating myocardium despite successful coronary artery bypass grafting at 4 wk post-revascularization

“…We have previously shown that maximal superoxide production is increased in isolated heart mitochondria from UCP-2 knock out compared with wild type mice 6 demonstrating UCP-2’s role in reducing oxidant stress. In addition, increased UCP-2 expression in swine hearts that underwent an ischemic preconditioning protocol during the second window of protection (SWOP) is associated with reduced reactive oxygen species 7 . In a swine model of hibernating myocardium, we have also shown that chronic ischemia leads to an increase in UCP-2 expression and reduced oxidant stress in mitochondria isolated from hibernating heart tissue 8 .…”