Abstract
Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.
Highlights
The cell survival program, termed ischemic preconditioning, evoked by transient nonlethal tissue ischemia, is evolutionarily conserved and is evident across multiple organs/tissues
Utilizing delayed ischemic preconditioning to identify transcriptional regulatory proteins governing mitochondrial biology and homeostasis, we previously demonstrated that the mitochondrial biogenesis regulatory protein peroxisome proliferator-activated receptor ␥ co-activator 1␣ is up-regulated in the preconditioned rat heart [3]
Using the rat heart delayed preconditioning protocol [3], we show that UCP2 and UCP3 gene transcripts are up-regulated (255 Ϯ 21 and 194 Ϯ 30%, respectively, p Ͻ 0.001) 4 h following ischemic preconditioning versus sham-operated controls (Fig. 1A)
Summary
Animal Experiments—The ischemic preconditioning trigger was achieved by conferring transient regional ischemia to the left coronary artery of Wistar rats as described previously [3]. H9c2 Cell Ischemic Preconditioning and Anoxia-Reoxygenation Protocols—RNA interference to deplete UCP2, UCP3, or both transcripts was performed in the rat H9c2 cell survival model [15]. This immortalized cell line is derived from embryonic BD1X rat heart tissue and was obtained from the American Type Culture Collection. The reperfusion phase lasted 2 h within the 5% CO2 incubator To precondition these cells, the anoxia-reoxygenation experiments were preceded by a 2-h anoxia followed by an 18-h recovery in normal growth medium prior to the anoxia-reoxygenation protocol. The cells underwent the transfection procedure 48 h prior to being inserted into the anoxic bags for exposure to the index anoxia and 2-h reperfusion protocol. Statistical Methods—Differences between data groups were evaluated for significance using Student’s t test or one-way analysis of variance. p Ͻ 0.05 was considered statistically significant, and data are expressed as mean Ϯ S.E
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