Antagonists of the Inhibitory Effect of Free Tubulin on VDAC Induce Oxidative Stress and...
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2985-Pos Board B677 Antagonists of the Inhibitory Effect of Free Tubulin on VDAC Induce Oxidative Stress and Mitochondrial Dysfunction David N. DeHart 1 , Monika Gooz 2 , Tatiana K. Rostovtseva 3 , Kely L. Sheldon 4 , John J. Lemasters 5 , Eduardo N. Maldonado 6 . 1 Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC, USA, 2 Medicine, Medical University of South Carolina, Charleston, SC, USA, 3 Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA, 4 W. Harry Feinstein Department of Molecular Microbiology and Immunology, John Hopkins University, Baltimore, MD, USA, 5 Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC, USA, 6 Drug Discovery & Biomedical Sciences; Hollings Cancer Center., Medical University of South Carolina, Charleston, SC, USA. Bakground: Mitochondrial membrane potential (DJ) and generation of reac- tive oxygen species (ROS) requires a respiratory chain fueled by the flux of metabolites into mitochondria through voltage dependent anion channels (VDAC). Free tubulin induces reversible blockage of VDAC both in vitro and in cells. Erastin, a small molecule lethal to cancer cells, antagonizes the inhibitory effect of free tubulin on VDAC and upregulates mitochondrial metabolism. Here, we hypothesized that erastin and "erastin-like" compounds open VDAC, increase mitochondrial metabolism and ROS formation, and activate JNK, which in turn cause mitochondrial dysfunction. Our AIM was to evaluate the effects of erastin/erastin-like compounds on DJ, NADH, ROS and JNK in intact cells. METHODS: DJ was assessed by confocal mi- croscopy of tetramethylrhodamine methylester (TMRM) fluorescence and ROS by chloromethyldichlorofluorescein (cmDCF) and MitoSOX Red fluorescence. Mitochondrial NADH autofluorescence was assessed by multi- photon microscopy. Total and phosphorylated JNK was determined by West- ern blotting. RESULTS: In lipid bilayers, erastin antagonized tubulin inhibition on VDAC. In HepG2 human hepatoma cells, erastin increased DJ by 46% and NADH by 30%. Mitochondrial hyperpolarization plateaued after 2 h. Subsequently depolarization occurred (3-4 h), suggesting mitochon- drial dysfunction. Erastin-like compounds X1 and X2, identified in a high- throughput screening, similarly caused mitochondrial hyperpolarization/ depolarization. Erastin also caused increases of DCF and Mitosox Red fluorescence beginning after ~30 min and reaching a maximum after 2 h. Additionally, erastin activated JNK (maximum pJNK at 60 min). JNK activa- tion and ROS formation both preceded mitochondrial depolarization. Conclu- sion: Erastin relieves tubulin-dependent inhibition of VDAC conductance, leading to mitochondrial hyperpolarization that increases ROS production with concomitant activation of the stress kinase JNK. These events, in turn, may induce the mitochondrial permeability transition, mitochondrial dysfunc- tion and ultimately cell death. 2986-Pos Board B678 Transient Mitochondrial Permeability Transition Pore Opening in Cardiac Myocytes During SR Ca Release Xiyuan Lu, Donald Bers. Pharamology, UC Davis, Davis, CA, USA. The opening of a high-conductance and long-lasting mitochondrial perme- ability transition pore (mPTP) induces uncoupling of respiration from ADP phosphorylation, and causes mitochondrial injury and cell death. How- ever, low-conductance and transient openings of mPTP may limit mitochon- drial calcium load and mediate mitochondrial reactive oxygen species (ROS) signaling. Transient openings of mPTP have been proposed, and could be cardioprotective, but evidence for this in cells is indirect and not thoroughly studied. To address the cellular mechanism, we measured mitochondrial [Ca] ([Ca]Mito) with Rhod-2 AM and membrane potential (Dcm) in isolated sin- gle permeablized myocytes during cyclical sarcoplasmic reticulum (SR) Ca release using 2-D confocal imaging where individual mitochondria can be seen. Rapid and transient decreases in both mitochondrial [Ca] and Dcm were observed during SR Ca release. The frequency of these candidate tran- sient mPTP openings increased at higher [Ca] and with H2O2 (1mM) expo- sure, but were typically observed in << 1% of individual mitochondria being imaged. This suggests that both Ca and ROS modulate transient pore openings. These [Ca]mito and Dcm oscillations and H2O2 effects were sensitive to mPTP inhibitor cyclosporine A (CsA, 10mM) and we conclude that they are mediated by transient mPTP openings and closings. The duration of these openings was 47 5 15 s. The size of the pore did not allow Rhod-2 or calcien (M.W. 600 Da) permeation, indicating that only small solutes can freely move across. Our data are consistent with the idea that rare transient mPTP openings allow individual mitochondrial Ca efflux, but with minimal perturbation of global average [Ca]mito or Dcm. These events may be an important physiological protective mechanism to limit [Ca]Mito overload. 2987-Pos Board B679 Spermine Selectively Inhibits High-Conductance but not Low- Conductance Mode of the Mitochondrial Permeability Transition Pore (MPTP) Pia A. Elustondo, Evgeny V. Pavlov. Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada. Spermine is a biological organic polymer that has four primary amino groups. It is involved in the regulation of transcription, cell signalling and enzymatic ac- tivity. It has also been demonstrated that polyamines inhibit calcium-induced high amplitude mitochondrial swelling, suggesting their involvement in regu- lation of the mitochondrial Permeability Transition Pore (mPTP). Here we further investigated the mechanisms of polyamine inhibition of calcium- induced, cyclosporine A (CSA) sensitive mPTP. Experiments were performed using isolated rat liver mitochondria resuspended in sucrose based recording solution. mPTP was induced by the addition of cal- cium to energized mitochondria. mPTP activation was measured by three inde- pendent approaches: 1) calcium release from the mitochondria detected with calcium green; 2) mitochondrial membrane depolarization using TMRM probe and 3) mitochondrial swelling measured as a decrease of light absorbance. In control experiments, in the presence of the specific inhibitor CSA, mPTP inhi- bition was observed by all three approaches. However, in the presence of sper- mine (0.02 mM to 0.2 mM) only mitochondrial swelling was inhibited, while membrane depolarization and calcium release were not affected. These results suggest that spermine inhibited high-conductance mode of mPTP, which opening is required for induction of mitochondrial swelling in sucrose based media. However spermine did not inhibit low-conductance mode mPTP. Our data are consistent with possible distinct regulation and/or nature of the different modes of mPTP. 2988-Pos Board B680 Control of Mitochondrial Ca 2D Uptake Threshold via the Micu1:Mcu Ratio Tu ¨nde Golena ´r 1 , Gyo ¨rgy Csorda ´s 1 , Gergo ¨ Szanda 2 , Cynthia Moffat 1 , Erin L. Seifert 1 , Andra ´s Spa ¨t 2 , Gyo ¨rgy Hajno ´czky 1 . 1 Pathology, Anatomy & Cell Biol., Thomas Jefferson University, Philadelphia, PA, USA, 2 Physiology, Semmelweis University, Budapest, Hungary. Mitochondrial Ca 2þ uptake mediated by the Ca 2þ uniporter plays important effector and modulator roles in cellular Ca 2þ signaling. The activation of the uniporter generally requires cytosolic [Ca 2þ ] ([Ca 2þ ] c ) >1mM; however, in certain cell types i is activated at much lower [Ca 2þ ] c . We have recently iden- tified the EF-hand protein MICU1 being responsible for both the relatively high threshold and positive cooperativity of the uniporter’s activation by [Ca 2þ ] c . Here we examined the idea that the cell-type-dependent differences in the [Ca 2þ ] c threshold for uniporter activation reflected differences in the relative availability of MICU1 to the pore protein MCU. First, we tested if decreasing the MICU1:MCU ratio via transient overexpression of MCU could lower the threshold for Ca 2þ uptake in HeLa cells. Simultaneous fluorescence imaging of [Ca 2þ ] c and [Ca 2þ ] in the mitochondrial matrix ([Ca 2þ ]m) confirmed a shorter coupling time between store-operated entry-derived [Ca 2þ ] c and [Ca 2þ ]m rises for the cells overexpressing MCU. Furthermore, upon permeabi- lization, elevated [Ca 2þ ]m baseline and robust enhancement of [Ca 2þ ]m response to small [Ca 2þ ] c rises was observed in MCU overexpressing cells. Importantly, overexpression of both MCU and MICU1 did not result sensitiza- tion of mitochondria to low [Ca 2þ ] c levels. Next, we compared MICU1 and MCU expression levels in H295R human adrenal carcinoma cells (threshold ~250nM) vs. Hela cells (threshold ~1mM) and in rat INS1 insulinoma cells (threshold ~190nM) vs. RBL2H3 mast cells (threshold ~1mM). Despite dis- playing a MICU1-deficient like [Ca 2þ ]m phenotype, H295R and INS1 cells their MICU1 expression was relatively strong and even more, their MI- CU1:MCU mRNA ratio was high. Thus, shifting the ratio of MICU1:MCU is an effective way of [Ca 2þ ] c threshold tuning for the uniporter in HeLa cells. However, threshold variances between cell types do not necessarily reflect MICU1:MCU differences but rather other alterations in the uniporter channel complex. 2989-Pos Board B681 Regulation of Mitochondrial Outer and Inner Membrane Fusion Coupling David Weaver, Xingguo Liu, Gyorgy Hajnoczky. Thomas Jefferson University, Philadelphia, PA, USA. Mitochondrial fusion is an essential process for maintaining cell health that en- tails the sequential mergers of outer membrane (OMM) and inner membrane Tuesday, February 18, 2014 591a
Transcript of Antagonists of the Inhibitory Effect of Free Tubulin on VDAC Induce Oxidative Stress and...
Tuesday, February 18, 2014 591a
2985-Pos Board B677Antagonists of the Inhibitory Effect of Free Tubulin on VDAC InduceOxidative Stress and Mitochondrial DysfunctionDavid N. DeHart1, Monika Gooz2, Tatiana K. Rostovtseva3,Kely L. Sheldon4, John J. Lemasters5, Eduardo N. Maldonado6.1Drug Discovery & Biomedical Sciences, Medical University of SouthCarolina, Charleston, SC, USA, 2Medicine, Medical University of SouthCarolina, Charleston, SC, USA, 3Eunice Kennedy Shriver National Instituteof Child Health and Human Development, Bethesda, MD, USA, 4W. HarryFeinstein Department of Molecular Microbiology and Immunology, JohnHopkins University, Baltimore, MD, USA, 5Drug Discovery & BiomedicalSciences and Biochemistry & Molecular Biology, Medical University ofSouth Carolina, Charleston, SC, USA, 6Drug Discovery & BiomedicalSciences; Hollings Cancer Center., Medical University of South Carolina,Charleston, SC, USA.Bakground: Mitochondrial membrane potential (DJ) and generation of reac-tive oxygen species (ROS) requires a respiratory chain fueled by the flux ofmetabolites into mitochondria through voltage dependent anion channels(VDAC). Free tubulin induces reversible blockage of VDAC both in vitroand in cells. Erastin, a small molecule lethal to cancer cells, antagonizesthe inhibitory effect of free tubulin on VDAC and upregulates mitochondrialmetabolism. Here, we hypothesized that erastin and "erastin-like" compoundsopen VDAC, increase mitochondrial metabolism and ROS formation, andactivate JNK, which in turn cause mitochondrial dysfunction. Our AIM wasto evaluate the effects of erastin/erastin-like compounds on DJ, NADH,ROS and JNK in intact cells. METHODS: DJ was assessed by confocal mi-croscopy of tetramethylrhodamine methylester (TMRM) fluorescence andROS by chloromethyldichlorofluorescein (cmDCF) and MitoSOX Redfluorescence. Mitochondrial NADH autofluorescence was assessed by multi-photon microscopy. Total and phosphorylated JNK was determined by West-ern blotting. RESULTS: In lipid bilayers, erastin antagonized tubulininhibition on VDAC. In HepG2 human hepatoma cells, erastin increasedDJ by 46% and NADH by 30%. Mitochondrial hyperpolarization plateauedafter 2 h. Subsequently depolarization occurred (3-4 h), suggesting mitochon-drial dysfunction. Erastin-like compounds X1 and X2, identified in a high-throughput screening, similarly caused mitochondrial hyperpolarization/depolarization. Erastin also caused increases of DCF and Mitosox Redfluorescence beginning after ~30 min and reaching a maximum after 2 h.Additionally, erastin activated JNK (maximum pJNK at 60 min). JNK activa-tion and ROS formation both preceded mitochondrial depolarization. Conclu-sion: Erastin relieves tubulin-dependent inhibition of VDAC conductance,leading to mitochondrial hyperpolarization that increases ROS productionwith concomitant activation of the stress kinase JNK. These events, in turn,may induce the mitochondrial permeability transition, mitochondrial dysfunc-tion and ultimately cell death.
2986-Pos Board B678Transient Mitochondrial Permeability Transition Pore Opening inCardiac Myocytes During SR Ca ReleaseXiyuan Lu, Donald Bers.Pharamology, UC Davis, Davis, CA, USA.The opening of a high-conductance and long-lasting mitochondrial perme-ability transition pore (mPTP) induces uncoupling of respiration fromADP phosphorylation, and causes mitochondrial injury and cell death. How-ever, low-conductance and transient openings of mPTP may limit mitochon-drial calcium load and mediate mitochondrial reactive oxygen species (ROS)signaling. Transient openings of mPTP have been proposed, and could becardioprotective, but evidence for this in cells is indirect and not thoroughlystudied. To address the cellular mechanism, we measured mitochondrial [Ca]([Ca]Mito) with Rhod-2 AM and membrane potential (Dcm) in isolated sin-gle permeablized myocytes during cyclical sarcoplasmic reticulum (SR) Carelease using 2-D confocal imaging where individual mitochondria can beseen. Rapid and transient decreases in both mitochondrial [Ca] and Dcmwere observed during SR Ca release. The frequency of these candidate tran-sient mPTP openings increased at higher [Ca] and with H2O2 (1mM) expo-sure, but were typically observed in << 1% of individual mitochondriabeing imaged. This suggests that both Ca and ROS modulate transientpore openings. These [Ca]mito and Dcm oscillations and H2O2 effectswere sensitive to mPTP inhibitor cyclosporine A (CsA, 10mM) and weconclude that they are mediated by transient mPTP openings and closings.The duration of these openings was 47 5 15 s. The size of the pore didnot allow Rhod-2 or calcien (M.W. 600 Da) permeation, indicating thatonly small solutes can freely move across. Our data are consistent withthe idea that rare transient mPTP openings allow individual mitochondrialCa efflux, but with minimal perturbation of global average [Ca]mito or
Dcm. These events may be an important physiological protective mechanismto limit [Ca]Mito overload.
2987-Pos Board B679Spermine Selectively Inhibits High-Conductance but not Low-Conductance Mode of the Mitochondrial Permeability Transition Pore(MPTP)Pia A. Elustondo, Evgeny V. Pavlov.Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada.Spermine is a biological organic polymer that has four primary amino groups. Itis involved in the regulation of transcription, cell signalling and enzymatic ac-tivity. It has also been demonstrated that polyamines inhibit calcium-inducedhigh amplitude mitochondrial swelling, suggesting their involvement in regu-lation of the mitochondrial Permeability Transition Pore (mPTP). Here wefurther investigated the mechanisms of polyamine inhibition of calcium-induced, cyclosporine A (CSA) sensitive mPTP.Experiments were performed using isolated rat liver mitochondria resuspendedin sucrose based recording solution. mPTP was induced by the addition of cal-cium to energized mitochondria. mPTP activation was measured by three inde-pendent approaches: 1) calcium release from the mitochondria detected withcalcium green; 2) mitochondrial membrane depolarization using TMRM probeand 3) mitochondrial swelling measured as a decrease of light absorbance. Incontrol experiments, in the presence of the specific inhibitor CSA, mPTP inhi-bition was observed by all three approaches. However, in the presence of sper-mine (0.02 mM to 0.2 mM) only mitochondrial swelling was inhibited, whilemembrane depolarization and calcium release were not affected.These results suggest that spermine inhibited high-conductance mode of mPTP,which opening is required for induction of mitochondrial swelling in sucrosebased media. However spermine did not inhibit low-conductance modemPTP. Our data are consistent with possible distinct regulation and/or natureof the different modes of mPTP.
2988-Pos Board B680Control of Mitochondrial Ca2D Uptake Threshold via the Micu1:McuRatioTunde Golenar1, Gyorgy Csordas1, Gergo Szanda2, Cynthia Moffat1,Erin L. Seifert1, Andras Spat2, Gyorgy Hajnoczky1.1Pathology, Anatomy & Cell Biol., Thomas Jefferson University,Philadelphia, PA, USA, 2Physiology, Semmelweis University, Budapest,Hungary.Mitochondrial Ca2þ uptake mediated by the Ca2þ uniporter plays importanteffector and modulator roles in cellular Ca2þ signaling. The activation of theuniporter generally requires cytosolic [Ca2þ] ([Ca2þ]c) >1mM; however, incertain cell types i is activated at much lower [Ca2þ]c. We have recently iden-tified the EF-hand protein MICU1 being responsible for both the relatively highthreshold and positive cooperativity of the uniporter’s activation by [Ca2þ]c.Here we examined the idea that the cell-type-dependent differences in the[Ca2þ]c threshold for uniporter activation reflected differences in the relativeavailability of MICU1 to the pore protein MCU. First, we tested if decreasingthe MICU1:MCU ratio via transient overexpression of MCU could lower thethreshold for Ca2þ uptake in HeLa cells. Simultaneous fluorescence imagingof [Ca2þ]c and [Ca2þ] in the mitochondrial matrix ([Ca2þ]m) confirmed ashorter coupling time between store-operated entry-derived [Ca2þ]c and[Ca2þ]m rises for the cells overexpressing MCU. Furthermore, upon permeabi-lization, elevated [Ca2þ]m baseline and robust enhancement of [Ca2þ]mresponse to small [Ca2þ]c rises was observed in MCU overexpressing cells.Importantly, overexpression of both MCU and MICU1 did not result sensitiza-tion of mitochondria to low [Ca2þ]c levels. Next, we compared MICU1 andMCU expression levels in H295R human adrenal carcinoma cells (threshold~250nM) vs. Hela cells (threshold ~1mM) and in rat INS1 insulinoma cells(threshold ~190nM) vs. RBL2H3 mast cells (threshold ~1mM). Despite dis-playing a MICU1-deficient like [Ca2þ]m phenotype, H295R and INS1 cellstheir MICU1 expression was relatively strong and even more, their MI-CU1:MCU mRNA ratio was high.Thus, shifting the ratio of MICU1:MCU is an effective way of [Ca2þ]cthreshold tuning for the uniporter in HeLa cells. However, threshold variancesbetween cell types do not necessarily reflect MICU1:MCU differences butrather other alterations in the uniporter channel complex.
2989-Pos Board B681Regulation of Mitochondrial Outer and Inner Membrane Fusion CouplingDavid Weaver, Xingguo Liu, Gyorgy Hajnoczky.Thomas Jefferson University, Philadelphia, PA, USA.Mitochondrial fusion is an essential process for maintaining cell health that en-tails the sequential mergers of outer membrane (OMM) and inner membrane
