Tyrosine Phosphorylation of Mitochondrial Ca2+ Uniporter Regulates Mitochondrial Ca2+ Uptake Wednesday, February 11, 2015 609a like ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA), are cytoprotective and inhibit cell death. The mechanisms associated with these distinct effects are not entirely clear. However, the effect of hydrophilic bile acids seems to be related with the blockage of a series of processes that converge on mitochondrial damage. Bax is a pro-apoptotic protein that belongs to the superfamily of the Bcl-2 proteins and is involved in mitochondrial pore formation. Submicellar concentrations of cytoprotective bile acids have been shown to modulate Bax concentration in mitochondria, suggesting that these molecules may interact directly with the protein. In this study, our objective was to evaluate the affinity of bile acids to recombinant Bax protein, making use of fluorescence spectroscopy (FRET and fluorescence anisotropy), as well as Fluorescence Correlation Spectroscopy (FCS). Our results show that the cytoprotective bile acids UDCA and TUDCA associate with recombinant Bax protein with high affinity, while the cytotoxic bile acid DCA only seems to be able to adsorb to the protein with much lower affinity. Notably, the bind- ing site for UDCA seems to be located in a hydrophobic pocket of the protein. This interaction could be responsible for the disruption of Bax translocation to the mitochondrial outer membrane in the presence of UDCA and/or TUDCA. Supported from FCT/Portugal (Projects PTDC/QUI-BIQ/119494/2010 and RECI/CTM-POL/0342/2012). T.S. and F.F. acknowledges FCT grants SFRH/BD/92398/2013 and SFRH/ BPD/64320/2009 3071-Pos Board B501 MAC Inhibitors Neutralize the Pro-Apoptotic Effects of Tbid Pablo M. Peixoto1, Oscar H. Teijido2, Laurent M. Dejean3, Evgeny Pavlov4, Bruno Antonsson5, Kathleen W. Kinnally4. 1Natural Sciences, Baruch College - CUNY, New York, NY, USA, 2National Institute of Child Health and Human Development, Baltimore, MD, USA, 3 Chemistry, California State University of Fresno, Fresno, CA, USA, 4 New York University College of Dentistry, New York, NY, USA, 5 Merck Serono, Geneva Research Center, Geneva, Switzerland. Since our initial characterization of the iMACs, different di-bromocarbazole derivatives with anti-apoptotic function have been developed and tested in several mouse models of brain injury and neurodegeneration [13-21]. Owing to the increased therapeutic potential of anti-apoptotic di-bromocarbazole de- rivatives, we sought to expand our knowledge of the mechanism of action of these small molecule inhibitors. We investigated the kinetics of MAC inhibi- tion in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and quantita- tive western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax oligomers in the outer membrane. A comparison of the inhibitory effects over channel conductance and cyto- chrome c release suggests that the iMACs interacted with both Bax and Bak with similar kinetics. Interestingly, wild type mitochondria were more suscep- tible to inhibition than the Bak or Bax knockouts. A quantitative western blot analysis showed that wild type mitochondria had lower steady state levels of Bak, which suggests an uneven stoichiometry of the MAC components. 3072-Pos Board B502 Tyrosine Phosphorylation of Mitochondrial Ca2D Uniporter Regulates Mitochondrial Ca2D Uptake Jin O-Uchi, Stephen Hurst, Jyotsna Mishra, Xiaole Xu, Bong Sook Jhun, Shey-Shing Sheu. Depariment of Medicine, Center for Translational Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA. Mitochondrial Ca2þ has a critical role for balancing cell survival and death. Ca2þ influx into mitochondrial matrix is mediated primarily by the mitochondrial cal- cium uniporter (MCU). However, the signaling pathways that regulate MCU channel functions via post-translational modifications of MCU are completely unknown. Here we show that adrenergic signaling induces MCU tyrosine phos- phorylation and accelerates mitochondrial Ca 2þ uptake in cardiac cells. Adren- ergic signaling induces activation of proline-rich tyrosine kinase 2 (Pyk2) and translocation into the mitochondrial matrix; enhancing the interaction between Pyk2 and MCU, which subsequently accelerates mitochondrial Ca2þ uptake via Pyk2-dependent MCU tyrosine phosphorylation. MCU contains 15 tyrosine res- idues (5 in the N-terminus, 0 in the pore-forming region, 4 in transmembrane do- mains and 6 in the C-terminus), which are conserved across all eukaryotic species. Among them, only 3 of these tyrosine residues (Y157 at N-terminus, Y288, and Y316 at C-terminus in mouse MCU) remained as potential phosphor- ylation candidate sites for protein tyrosine kinases using phosphorylation predic- tion programs. We mutated these tyrosine residues to phenylalanine and generated non-phosphorylation mimetic MCU mutants (MCU-YFs). We confirmed that only two tyrosine sites were phosphorylated in response to adren- ergic stimulation in situ using cell lines stably expressing MCU-YFs. In addi- tion, overexpression of these MCU-YFs failed to increase mitochondrial Ca 2þ uptake in response to cytosolic Ca 2þ elevation by thapsigargin, whereas wild- type MCU transfection dramatically accelerates mitochondrial Ca2þ uptake compared to non-transfected cells. In summary, MCU contains Pyk2-specific phosphorylation site(s) and Pyk2-dependent tyrosine phosphorylation of MCU can modulate its channel functions and regulate mitochondrial Ca 2þ uptake. 3073-Pos Board B503 Cardioprotective Roles of Neuronal Ca 2D Sensor-1 during Stress Tomoe Y. Nakamura-Nishitani1, Shu Nakao1, Shigeo Wakabayashi2. 1Molecular Physiology, Natl.Cer.Cardiovasc.Ctr., Suita, Japan, 2Cardiac Physiology, Natl.Cer.Cardiovasc.CtrWakabayshi, Suita, Japan. Dysregulation of Ca2þ homeostasis in cardiomyocytes often results in heart fail- ure. Identifying molecular targets that regulate cardiomyocyte survival is of ther- apeutic importance. Neuronal Ca 2þ -sensor-1 (NCS-1) is an EF-hand Ca 2þ - binding protein, which is important for excitable cell functions. We previously found that NCS-1-deficient (Ncs1�/�) mice had excess neonatal mortality (Circ. Res.2011).The aim ofthe present study isto examinewhether NCS-1plays beneficial roles in cardiac survival during stress and the possible mechanisms un- derlying these effects. Neonatal mouse ventricular myocytes orwhole hearts from wild-type (WT) and Ncs1 �/� mice were subjected to stressors, and the resistance to stress was evaluated. Ncs1 �/� mouse hearts were more susceptible to stress induced by oxidative impairment and ischemia-reperfusion injury. Stress- inducedactivationofphosphatidylinositol3-kinase (PI3K)/Akt signaling, a major survival pathway, was substantially reduced in the Ncs1�/�group, and overex- pressionofNCS-1 ortheconstitutiveactive formofAkt increased the survivalrate of Ncs1 �/� myocytes. Cellular ATP levels, as well as mitochondrial respiration rates (both basal and maximal O2 consumption) were significantly depressed in Ncs1 �/� myocytes; especially with oxidative stress. Furthermore, intracellular Ca2þ handling was more easily dysregulated in stressed Ncs1�/� myocytes than WT myocytes. Since NCS-1 levels were increased by stress, the data suggest that NCS-1 is a survival-promoting factor, which is upregulated by stress stimuli. Interestingly, however,supra-physiological NCS-1 expression was toxic to cells. Taken together, our data suggest that moderate NCS-1 expression during stress promotes cardiomyocyte survival by maintaining proper Ca 2þ handling, which is required for activation of Akt survival pathways and mitochondrial function. 3074-Pos Board B504 Initiation of Electron Transport Activity and a Decrease of Oxidative Stress Occur Simultaneously during Embryonic Heart Development Gisela Beutner, George A. Porter, Jr. Pediatrics, University of Rochester Medical Center, Rochester, NY, USA. Mitochondria in early embryonic hearts are not thought to produce ATP, yet they do produce reactive oxygen species (ROS) that regulate myocyte differen- tiation. To assess changes in ATP and ROS generation in the developing heart, we measured mitochondrial oxygen consumption, the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC), ETC supercomplex assem- bly, and ROS in embryonic mouse hearts. At embryonic day (E) 9.5, mitochon- drial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the ETC complexes are present. We show that Cx-1 is able to accept electrons from the Krebs cycle, but enzyme assays that specifically mea- sure electron flow to ubiquinone or Cx-3 show no activity at this early embry- onic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx �1, �3, and �4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochon- dria. In contrast, generation of reactive oxygen species (ROS), as measured with Amplex Red, is high at E9.5 and drops significantly by E11.5, coinciding with activation of the ETC. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in a decrease of ROS generation and an increase in OXPHOS due to the activation of Cx-1 and the formation of supercomplexes. 3075-Pos Board B505 The Stoichiometry between MICU1 and MCU Determines the Different Mitochondrial Ca 2D Uptake Phenotypes in Heart and Liver Melanie Paillard, György Csordás, Tünde Golenár, Cynthia Moffat, Erin Seifert, György Hajnóczky. MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, PA, USA. Mitochondrial Ca2þ uptake is central to oxidative metabolism and cell death signaling. The first clues to its molecular mechanism have emerged from the recent identification of the mitochondrial Ca 2þ uniporter’s pore forming protein (MCU) as well as its regulators. Among the regulators, MICU1 shows striking MAC Inhibitors Neutralize the Pro-Apoptotic Effects of Tbid Tyrosine Phosphorylation of Mitochondrial Ca2+ Uniporter Regulates Mitochondrial Ca2+ Uptake Cardioprotective Roles of Neuronal Ca2+ Sensor-1 during Stress Initiation of Electron Transport Activity and a Decrease of Oxidative Stress Occur Simultaneously during Embryonic Heart De ... The Stoichiometry between MICU1 and MCU Determines the Different Mitochondrial Ca2+ Uptake Phenotypes in Heart and Liver