Uncoupling Proteins 2 and 3 Function in Concert to Augment Tolerance to Cardiac Ischemia

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. 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. The cell survival program, termed ischemic preconditioning, evoked by transient nonlethal tissue ischemia, is evolutionarily conserved and is evident across multiple organs/tissues. The signal transduction networks engendering this phenomenon are well described (1Yellon D.M. Downey J.M. Physiol. Rev. 2003; 83: 1113-1151Crossref PubMed Scopus (869) Google Scholar); however, functional cellular modifications conferring ischemia tolerance are less tangible. Interestingly, the invariable metabolic signature of preconditioned tissue is enhanced capacity to restore mitochondrial function and ATP production following an ischemic insult (2Opie L.H. Sack M.N. J. Mol. Cell. Cardiol. 2002; 34: 1077-1089Abstract Full Text PDF PubMed Google Scholar). The mechanistic links between the molecular adaptations of the mitochondrion and the preconditioning cell survival program require further characterization. Further credence to the importance of mitochondrial adaptation to preconditioning-induced cell survival has been shown by improved mitochondrial respiratory and bioenergetic recovery following anoxia and reoxygenation in mitochondria extracted from ischemic, genomic, and pharmacologic preconditioned hearts (3McLeod C.J. Jeyabalan A.P. Minners J.O. Clevenger R. Hoyt Jr., R.F. Sack M.N. Circulation. 2004; 110: 534-539Crossref PubMed Scopus (57) Google Scholar, 4McCarthy J. McLeod C.J. Minners J. Essop M.F. Ping P. Sack M.N. J. Mol. Cell Cardiol. 2005; 38: 697-700Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 5Ozcan C. Bienengraeber M. Dzeja P.P. Terzic A. Am. J. Physiol. 2002; 282: H531-H539Crossref PubMed Scopus (197) Google Scholar). In addition, preconditioned mitochondria generate less reactive oxygen species (ROS) 3The abbreviations used are: ROS, reactive oxygen species; 2-MPG, 2-mercaptopropionyl glycine; DCF, dichlorofluorescein; RNAi, RNA interference; 7-AAD, 7-amino-actinomycin D.3The abbreviations used are: ROS, reactive oxygen species; 2-MPG, 2-mercaptopropionyl glycine; DCF, dichlorofluorescein; RNAi, RNA interference; 7-AAD, 7-amino-actinomycin D. at reoxygenation than do nonpreconditioned mitochondria (5Ozcan C. Bienengraeber M. Dzeja P.P. Terzic A. Am. J. Physiol. 2002; 282: H531-H539Crossref PubMed Scopus (197) Google Scholar, 6Oshima Y. Fujio Y. Nakanishi T. Itoh N. Yamamoto Y. Negoro S. Tanaka K. Kishimoto T. Kawase I. Azuma J. Cardiovasc. Res. 2005; 65: 428-435Crossref PubMed Scopus (136) Google Scholar, 7Glantz L. Avramovich A. Trembovler V. Gurvitz V. Kohen R. Eidelman L.A. Shohami E. Exp. Neurol. 2005; 192: 117-124Crossref PubMed Scopus (57) Google Scholar). The broad molecular regulatory programs underpinning mitochondrial adaptations in preconditioning is further underscored by additional modulations in nuclear-encoded proteins modulating mitochondrial function during preconditioning (8Yamashita N. Hoshida S. Taniguchi N. Kuzuya T. Hori M. J. Mol. Cell Cardiol. 1998; 30: 1181-1189Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 9Ardehali H. Chen Z. Ko Y. Mejia-Alvarez R. Marban E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11880-11885Crossref PubMed Scopus (188) Google Scholar). 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 γ co-activator 1α is up-regulated in the preconditioned rat heart (3McLeod C.J. Jeyabalan A.P. Minners J.O. Clevenger R. Hoyt Jr., R.F. Sack M.N. Circulation. 2004; 110: 534-539Crossref PubMed Scopus (57) Google Scholar). Since peroxisome proliferator-activated receptor γ co-activator 1α up-regulates mitochondrial uncoupling proteins (10St-Pierre J. Lin J. Krauss S. Tarr P.T. Yang R. Newgard C.B. Spiegelman B.M. J. Biol. Chem. 2003; 278: 26597-26603Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar), we reasoned that this family of inner mitochondrial membrane carrier proteins may be functional components of the delayed preconditioning program. In this report, we examine the molecular and biochemical regulation of cardiac enriched inner mitochondrial membrane uncoupling proteins (UCP2 and UCP3) in delayed preconditioning and as a component of innate cellular tolerance to anoxia-reoxygenation. We demonstrate that UCP2 and -3 are up-regulated by the delayed ischemic preconditioning program. We show that these regulatory events are ROS-inducible and appear to attenuate subsequent anoxia-reoxygenation-mediated mitochondrial ROS production. Using RNA interference in rat heart H9c2 cells, we functionally demonstrate the individual and combined requirement of UCP2 and UCP3 in the preconditioning program and in innate cellular tolerance against anoxia-reoxygenation-provoked cell death. Animal Experiments—The ischemic preconditioning trigger was achieved by conferring transient regional ischemia to the left coronary artery of Wistar rats as described previously (3McLeod C.J. Jeyabalan A.P. Minners J.O. Clevenger R. Hoyt Jr., R.F. Sack M.N. Circulation. 2004; 110: 534-539Crossref PubMed Scopus (57) Google Scholar). Here, the heart was subjected to five episodes of 3 min of ischemia coupled to 5 min of reperfusion. Sham operations involved encircling the left coronary artery without tightening the suture. Since inhibition of antioxidant signaling abolishes the preconditioning program, 2-mercaptopropionyl glycine (2-MPG) was administered to an additional group of rats (0.42 mg/kg/min 2-MPG), beginning 15 min before preconditioning ischemia and continued until 1 h after the last occlusion as an additional control group. Following chest closure, animals were allowed to recover. Hearts were then extracted at 4 h for RNA extraction and at 24 h following the procedure for (i) mitochondrial isolation (11Sordahl L.A. Besch Jr., H.R. Allen J.C. Crow C. Lindenmayer G.E. Schwartz A. Methods Achiev. Exp. Pathol. 1971; 5: 287-346PubMed Google Scholar) and analysis and (ii) Western blot analysis. Animal experiments were approved by the NHLBI Animal Care and Use Committee. Quantitative Real Time PCR and Western Blot Analysis—Total RNA samples were treated with DNase I (Invitrogen) and reverse-transcribed using oligo(dT)12-18, SuperScript™ (Invitrogen). Real time quantitative PCR analysis was performed using an MJResearch Opticon II and SYBR Green (Stratagene) as the fluorophore. Standards were prepared using serial dilutions of cDNA in the range of 1 million to 1 copy per reaction, and gene expression was quantified by comparison with the standard curve. All gene expression was normalized to 18 S rRNA and glyceraldehyde-3-phosphate dehydrogenase as internal controls. UCP2 antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Chemicon were used for immunoblot analysis. 100 μg of crude whole cell protein extract was run using standard SDS-PAGE techniques, and the secondary antibody was conjugated to horseradish peroxidase. The antigen-antibody reaction was detected using enhanced chemiluminescence reagents (Amersham Biosciences), and β-actin levels and Ponceau staining (Sigma) were measured to confirm protein integrity and equal loading. Mitochondrial Isolation and Respiratory Analysis—Mitochondria were isolated (11Sordahl L.A. Besch Jr., H.R. Allen J.C. Crow C. Lindenmayer G.E. Schwartz A. Methods Achiev. Exp. Pathol. 1971; 5: 287-346PubMed Google Scholar) and respiratory function evaluated using fiber-optic oxygen spectrophotometry (Instech Laboratories). Mitochondria (0.35 mg·ml-1) were resuspended in a standard sucrose-Tris assay medium (pH 7.2), and basal respiratory rates were calculated in the presence of glutamate, malate, and ADP (5, 5, and 500 nm, respectively). Basal proton leak kinetics were evaluated with the addition of oligomycin (1 μg·ml-1), whereas the inducible proton leak measurements were performed in the presence of succinate (4 mm) and oligomycin (1 μg·ml-1). GDP (500 μm) was subsequently added to assess the response to this UCP inhibitor (12Jaburek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 13Zackova M. Skobisova E. Urbankova E. Jezek P. J. Biol. Chem. 2003; 278: 20761-20769Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Mitochondrial Anoxia-Reoxygenation and Hydrogen Peroxide Production—Mitochondria (0.35 mg·ml-1) were resuspended in the assay buffer (containing excess ADP, 5 mm glutamate, and 5 mm malate) in a sealed, stirred chamber. Anoxia was induced in ≤3 min via consumption of available oxygen in the air-tight chamber. Anoxia was for The was then with and hydrogen peroxide was subsequently using the assay 2004; PubMed Scopus Google Scholar). The of production was calculated by the in in the presence of this mg·ml-1) coupled to the of horseradish (4 with 2004; PubMed Scopus Google Scholar). The was against were run H9c2 Cell Preconditioning and Anoxia-Reoxygenation interference to or both transcripts was performed in the rat H9c2 cell survival M. C. Terzic A. J. Mol. Cell Cardiol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This cell is derived from rat heart tissue and was from the were in a and medium to The were then subjected to anoxia by The h of this before and with The 2 h the To precondition these cells, the anoxia-reoxygenation experiments were by a anoxia by an recovery in medium to the anoxia-reoxygenation to rat UCP2 and UCP3 were by H9c2 were with these using (Invitrogen). The of gene was using time quantitative PCR for UCP2 and UCP3 and by Western blot analysis for The control RNAi is by and has with rat or The the procedure h to the for to the anoxia and the preconditioning a preconditioning trigger was h following that anoxia-reoxygenation were performed h following The ischemia for the preconditioning was h and to h for nonpreconditioned The were in a for cellular viability or ROS for the of production between were evaluated for using or analysis of was and are as Using the rat heart delayed preconditioning (3McLeod C.J. Jeyabalan A.P. Minners J.O. Clevenger R. Hoyt Jr., R.F. Sack M.N. Circulation. 2004; 110: 534-539Crossref PubMed Scopus (57) Google Scholar), we show that UCP2 and UCP3 gene transcripts are up-regulated and 4 h following ischemic preconditioning Interestingly, this gene regulatory is by inhibition of delayed preconditioning with the antioxidant and preconditioning H. A. Y. Zhang R. Am. J. Physiol. 2002; 282: PubMed Scopus Google Scholar). In parallel with gene UCP2 protein levels are up-regulated 24 h following the ischemic trigger The UCP3 is less than UCP2 in the rat heart. This for to protein levels of To the of these inner mitochondrial carrier proteins respiratory mitochondria were isolated from rat hearts 24 h following ischemic preconditioning and Basal mitochondrial oxygen consumption 3 and are between preconditioned and nonpreconditioned and in basal ROS levels were measured not Interestingly, UCP2 and -3 been shown to be during basal but demonstrate a capacity to a mitochondrial proton leak in response to ROS signaling J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Zhang L. J. J. 2003; PubMed Scopus Google Scholar). Mitochondrial ROS was via in with inhibition using oligomycin D. J. S. A. S. J.C. 2002; PubMed Scopus Google Scholar, M. M. J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Here, preconditioned mitochondria a 15 greater inducible proton leak compared with control mitochondria this enhanced inducible leak is by the UCP GDP (12Jaburek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 13Zackova M. Skobisova E. Urbankova E. Jezek P. J. Biol. Chem. 2003; 278: 20761-20769Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) Since of an inducible proton leak following delayed preconditioning be with an of mitochondrial ROS in response to we evaluated the of at The delayed preconditioned mitochondria a reduction in production at reoxygenation following min of in anoxia compared with control mitochondria as measured preconditioned mitochondria exhibit a proton oligomycin in nonpreconditioned nonpreconditioned nonpreconditioned mitochondria. in a In to the functional requirement of UCP2 and UCP3 in modulating tolerance to preconditioning and we RNA interference to UCP2 and -3 in rat H9c2 cells. In parallel with the rat UCP2 is the uncoupling protein in H9c2 and is in excess of greater than the UCP3 not RNAi in a reduction in UCP2 and an reduction in UCP3 and a combined depletion in response to RNAi of both as measured by time quantitative PCR UCP2 protein levels were to of expression was from 24 to h following RNAi and control RNAi were in equal in and cell viability was by using the RNA not cellular viability a not The H9c2 exhibit preconditioning with viability viability in nonpreconditioned in response to the anoxia-reoxygenation insult depletion of UCP2 abolished whereas UCP3 depletion partially abolished the phenotype. however, was significantly greater than non-preconditioned but significantly less than that in control RNAi preconditioned the preconditioning trigger modifications in cellular tolerance to subsequent we then evaluated genomic depletion of the cardiac enriched anoxia-reoxygenation following h of anoxia and 2 h of was from but viability with UCP3 depletion to of viability following UCP2 and combined UCP2 and UCP3 depletion compared with RNAi 5, and The cell viability from in the RNAi to viability with UCP3 depletion not to following UCP2 RNAi to following the UCP2 and UCP3 depletion 5, and depletion of UCP2 and the combined UCP2 and UCP3 depletion cell in response to anoxia-reoxygenation is via ROS production. To assay ROS the of was measured using for viability The of with cell The ROS was in with RNAi, and the ROS was in in both UCP2 and UCP3 5, and Interestingly, in the viability was the were with 1 mm the anoxia-reoxygenation In and ROS production was attenuated with RNA viability and augmented ROS production following anoxia-reoxygenation in H9c2 cells. with the anti-oxidant the UCP2 phenotype. of viable cells. derived from ROS in for viability via of are shown as All of these experiments were performed at in control RNAi results show that the delayed preconditioning program up-regulates UCP2 and UCP3 in the rat heart. This genomic regulation is with an inducible mitochondrial proton leak and an augmented capacity to production. In parallel, the preconditioning is abolished following UCP2 RNAi depletion and partially by UCP3 depletion in H9c2 cells. Functional characterization of these inner mitochondrial membrane proteins in the rat heart-derived myoblasts that depletion of UCP2 or both UCP2 and UCP3 is with a cellular tolerance to anoxia-reoxygenation and is with production of this to anoxia-reoxygenation is by the administration of the antioxidant these a role of the cardiac enriched uncoupling proteins in innate ischemia tolerance and demonstrate that these inner mitochondrial membrane carrier proteins are as components of the delayed ischemic preconditioning cell survival program. following the ischemic preconditioning the heart with nonpreconditioned hearts (3McLeod C.J. Jeyabalan A.P. Minners J.O. Clevenger R. Hoyt Jr., R.F. Sack M.N. Circulation. 2004; 110: 534-539Crossref PubMed Scopus (57) Google Scholar). 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The reduction in ROS in the isolated preconditioned mitochondria both the of the uncoupling proteins and antioxidant as are by the preconditioning program. gene depletion do the of the UCP in ROS this an regulatory program these inner mitochondrial membrane proteins are up-regulated and functionally by ROS to subsequent mitochondrial ROS production. are with the evolutionarily conserved function of the uncoupling proteins to mitochondrial ROS D. H. C. S. E. M. R. D. S. D. PubMed Scopus Google Scholar, M. K. S. R.F. T. M. K. T. 2003; PubMed Scopus Google Scholar) as in and (12Jaburek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, M. M. J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, N. C. Res. 2003; PubMed Scopus Google Scholar, J. Biol. 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The in this that uncoupling proteins in the mitochondria are by innate cell survival signaling as by The RNA interference demonstrate that the mitochondrial uncoupling proteins are necessary components of cellular tolerance to and the delayed preconditioning program. we that these of the mitochondria function as a cellular cellular and cell be that the of the mitochondrial uncoupling protein are and a of metabolic and signaling S. Zhang Rev. Mol. Cell Biol. 2005; PubMed Scopus Google Scholar, P. J.C. A. M. Green K. J. 2004; PubMed Scopus Google Scholar, M. S. P. Garlid K.D. Jezek P. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) that not been in this but may to role in cellular In conclusion, we previously demonstrated that isolated mitochondria following of the delayed preconditioning cell survival program are of anoxia-reoxygenation (3McLeod C.J. Jeyabalan A.P. Minners J.O. Clevenger R. Hoyt Jr., R.F. Sack M.N. Circulation. 2004; 110: 534-539Crossref PubMed Scopus (57) Google Scholar). we demonstrate that this mitochondrial is with an of the cardiac enriched UCP2 and UCP3 In addition, preconditioned mitochondria exhibit a ROS-inducible uncoupling of as quantified by in response to GDP preconditioned mitochondria shown enhanced capacity to attenuate ROS following anoxia-reoxygenation. The depletion of the UCP2 and UCP3 in mitochondria in further that these inner mitochondrial membrane proteins are a necessary component of cellular antioxidant defense be by ROS UCP2 and UCP3 as cytoprotective proteins that are both and induced in response to ischemic cytoprotective parallel the expression of these mitochondrial uncoupling protein in that UCP2 depletion has greater to anoxia than does UCP3 UCP2 and UCP3 are shown to be necessary components of innate cellular antioxidant a functional role of the uncoupling proteins in tissue and the mitochondrial uncoupling proteins as for the of ischemic We D. of Garlid and S. of for and and for of the