The long-term goals of this project are to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardioprotection; 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection and 3) to understand changes in cytosolic and mitochondrial signaling involved in cardioprotection and cell death. It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. PC is known to involve nitric oxide signaling. Recent data have shown that cardioprotection can result in the import of specific proteins into the mitochondria in a process that involves heat shock protein 90 (HSP90) and is blocked by geldanamycin (GD), a HSP90 inhibitor. To test the hypothesis that an alteration in mitochondrial import is a more widespread feature of cardioprotection, in this study, we used a broad-based proteomics approach to investigate changes in the mitochondrial proteome following cardioprotection induced by inhibition of glycogen synthase kinase (GSK)-3. Mitochondria were isolated from control hearts, and hearts were perfused with the GSK inhibitor SB 216763 (SB) for 15 min before isolation of mitochondria. Mitochondrial extracts from control and SB-perfused hearts were labeled with isotope tags for relative and absolute quantification (iTRAQ), and differences in mitochondrial protein levels were determined by mass spectrometry. To test for the role of HSP90-mediated protein import, hearts were perfused in the presence and absence of GD for 15 min before perfusion with SB followed by mitochondrial isolation and iTRAQ labeling. We confirmed that treatment with GD blocked the protection afforded by SB treatment in a protocol of 20 min of ischemia and 40 min of reperfusion. We found 16 proteins that showed an apparent increase in the mitochondrial fraction following SB treatment. GD treatment significantly blocked the SB-mediated increase in mitochondrial association for five of these proteins, which included annexin A6, vinculin, and pyruvate kinase. We also found that SB treatment resulted in a decrease in mitochondrial content of eight proteins, of which all but two are established mitochondrial proteins. To confirm a role for mitochondrial import versus a change in protein synthesis and/or degradation, we measured changes in these proteins in whole cell extracts. Taken together, these data show that SB leads to a remodeling of the mitochondrial proteome that is partially GD sensitive. We were also interested in examining the physiological role of cyclophilin D. Isolated mitochondria from mice deficient in cyclophilin D (CypD-/-) are less sensitive to Ca2+-induced opening of the mitochondrial permeability transition (MPT) in vitro. Thus, the lack of CypD enables heart mitochondria to take up more Ca2+ before undergoing the MPT. We hypothesize that the MPT serves as a Ca2+-safety valve that can open to release excess Ca2+, but not necessarily result in death. If the MPT is blocked in CypD-/- mice, we hypothesize that matrix Ca2+ (Ca2+m) would be higher in CypD-/- mice compared to WT and this would activate Ca2+-sensitive NADH dehydrogenases (e.g., pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH)), which would in turn, alter oxidative metabolism and increase oxygen consumption. Consistent with this, we found altered expression levels of PDH E1 subunit and the alpha-KGDH E2 subunit in CypD-/- hearts using 2D DIGE proteomics. Therefore, these results demonstrate that the loss of a MPT component, CypD, results in physiological flux changes in the Krebs cycle and oxidative metabolism that are consistent with increased Ca2+m. As described above. mice lacking cyclophilin D (CypD-/-), a mitochondrial chaperone protein, have altered cardiac metabolism. As acetylation has been shown to regulate metabolism, we tested whether changes in protein acetylation might play a role in these metabolic changes in CypD-/- hearts. To identify changes in lysine-acetylated proteins and map acetylation sites following ablation of CypD, we subjected tryptic digests of isolated cardiac mitochondria from WT and CypD-/- mice to immunoprecipitation using agarose beads coupled to anti-acetyl lysine antibodies followed by mass spectrometry. We used label-free analysis for the relative quantification of the 875 common peptides that were acetylated in WT and CypD-/- samples and found 11 peptides (10 proteins) decreased and 96 peptides (48 proteins) increased in the CypD-/- samples. We found increased acetylation of proteins in fatty acid oxidation and branched-chain amino acid metabolism. To evaluate whether this increase in acetylation might play a role in the inhibition of fatty acid oxidation that was previously reported in CypD-/- hearts, we measured the activity of L-3-hydroxyacyl-CoA dehydrogenase (LCHAD), which was acetylated in the CypD-/- hearts. Consistent with the hypothesis, LCHAD activity was inhibited by approximately 50% compared to the WT mitochondria. These results implicate a role for CypD in modulating protein acetylation. Taken together, these results suggest that ablation of CypD leads to changes in the mitochondrial acetylome, which may contribute to altered mitochondrial metabolism in CypD-/- mice.

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Murphy, Elizabeth; Pan, Xin; Nguyen, Tiffany et al. (2014) Unresolved questions from the analysis of mice lacking MCU expression. Biochem Biophys Res Commun 449:384-5
Yano, Toshiyuki; Ferlito, Marcella; Aponte, Angel et al. (2014) Pivotal role of mTORC2 and involvement of ribosomal protein S6 in cardioprotective signaling. Circ Res 114:1268-80
Beca, Sanja; Ahmad, Faiyaz; Shen, Weixing et al. (2013) Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart. Circ Res 112:289-97
Evangelista, Alicia M; Kohr, Mark J; Murphy, Elizabeth (2013) S-nitrosylation: specificity, occupancy, and interaction with other post-translational modifications. Antioxid Redox Signal 19:1209-19
Chess, David J; Billings, Eric; Covian, Raul et al. (2013) Optical spectroscopy in turbid media using an integrating sphere: mitochondrial chromophore analysis during metabolic transitions. Anal Biochem 439:161-72
Menazza, Sara; Wong, Renee; Nguyen, Tiffany et al. (2013) CypD(-/-) hearts have altered levels of proteins involved in Krebs cycle, branch chain amino acid degradation and pyruvate metabolism. J Mol Cell Cardiol 56:81-90
Lin, Jeffrey; Steenbergen, Charles; Murphy, Elizabeth et al. (2009) Estrogen receptor-beta activation results in S-nitrosylation of proteins involved in cardioprotection. Circulation 120:245-54
Steenbergen, Charles; Das, Samarjit; Su, Jason et al. (2009) Cardioprotection and altered mitochondrial adenine nucleotide transport. Basic Res Cardiol 104:149-56
Murphy, Elizabeth; Eisner, David A (2009) Regulation of intracellular and mitochondrial sodium in health and disease. Circ Res 104:292-303
Sack, Michael N; Murphy, Elizabeth; Schulz, Rainer (2009) The regulation and control of mitochondrial homeostasis in changing cardiac tolerance to ischemia-reperfusion injury: a focused issue. Basic Res Cardiol 104:111-2

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