ER-Mitochondrial Ultrastructure in the Liver of Normal and Ethnaol-Fed Rats 216a Monday, February 4, 2013 activates a novel, calcium-independent, PKCa-dependent signaling pathway, which results in mitochondrial depolarization. As a result, mitochondrial dys- function is likely to be a key contributor to the pathophysiology of gas embo- lism injury. Further, this connection between the endothelial surface layer and endothelial mitochondria may also play an important role in vascular homeo- stasis and disease. 1105-Plat Calcium-Induced ROS Generation during Ischemia Triggers mPTP- Dependent Cell Death during Reperfusion Lea K. Seidlmayer, Vanessa V. Juettner, Lothar A. Blatter, Elena N. Dedkova. Rush University Medical Center, Chicago, IL, USA. Impairment of mitochondrial function is a central event of ischemia- reperfusion (I/R) injury leading to tissue damage and cell death. We studied the relationship between mitochondrial Ca 2þ overload, reactive oxygen species (ROS) generation, opening of the mitochondrial permeability transition pore (mPTP) and cell death in rabbit ventricular myocytes exposed to simulated I/R. Changes in mitochondrial Ca2þ ([Ca2þ]m) were measured with X-Rhod- 1, ROS generation with Mito-Sox Red, mPTP opening as mitochondrial calcein red release, and cell death as lactate dehydrogenase release. I/R was induced by exposing cells to glucose-free Tyrode solution containing 20 mM 2-deoxyglu- cose and 2 mM NaCN, pH 6.4, followed by superfusion with standard Tyrode solution. No cell death was observed after 20 min of ischemia despite a signif- icant increase in [Ca2þ]m, ROS and mPTP opening, however cell death in- creased significantly after 15 min of reperfusion. The Ca2þ uniporter blocker Ru360 partially prevented [Ca 2þ ]m increase and completely abolished ROS generation and cell death when applied during I/R, however application of Ru360 only during reperfusion did not protect from cell death. Scavenging ROS with superoxide dismutase mimetic MnTBAP or antioxidant Trolox pre- vented reperfusion-induced cell death. Blocking mPTP during ischemia by cyclosporine A or depletion of mitochondrial inorganic polyphosphate did not provide protection against cell death during reperfusion, but instead led to increased mitochondrial ROS accumulation. However, inhibiting mPTP opening during reperfusion with cyclosporine A attenuated cell death. We con- clude that mPTP-dependent cell death during reperfusion is mediated by Ca2þ- dependent ROS generation during ischemia. Moreover, our data suggest that mPTP opening during ischemia could serve as ROS escape pathway from mitochondria and thereby attenuate mitochondrial ROS accumulation and ROS-mediated cell damage during subsequent reperfusion. 1106-Plat Dynamic Measurement of Ca 2D -Induced Changes in Organelle-Specific Redox Microdomains David M. Booth1, Balázs Enyedi2, Miklós Geiszt2, Péter Várnai2, György Hajnóczky1. 1 Thomas Jefferson University, Philadelphia, PA, USA, 2 Semmelweis University, Budapest, Hungary. Multiple interactions between Ca 2þ -signalling and reactive oxygen species (ROS) production are thought to exist and be of both physiological and patho- physiological relevance. However, until recently, the study of ROS in the con- text of Ca2þ-signalling has been hampered by ROS probes limited in both specificity and targeting. To measure ROS in discrete subcellular domains, we compared the genetically-encoded ratiometric H2O2 sensor HyPer with the redox de-sensitized derivative SypHer to control for non-specific changes. These probes, targeted to the ER lumen, ER membrane (cytosolic face), cyto- sol, mitochondrial matrix and outer membrane (OMM) and the IP3-receptor, are positioned to assess redox changes at a local level. In resting conditions, the ER lumen showed >3-fold increased HyPer ratio compared to the outside of the ER membrane, bulk cytosol and mitochondrial matrix and OMM. In the ER lumen, IP3-linked Ca 2þ -mobilization induced a prounounced, downward shift in HyPer ratio, whereas little or no change was detected in other compart- ments. In permeabilized cells, mitochondrial Ca2þ-overload was accompanied by substantial increase in H2O2 detected in the matrix and at the OMM. These data demonstrate that redox environments within individual cellular compart- ments constitute a complex and dynamic interrelationship with the concentra- tion of free Ca 2þ . The ER lumen is highly oxidized and exhibits profound decreases in H2O2 concommitant with Ca 2þ -release. Interestingly, these changes are not transmitted to the outer leaflet of the ER membrane or to other compartments. Mitochondria resist redox changes under moderate stimulation, however, during mitochondrial Ca2þ-overload and collapsed membrane poten- tial, strong H2O2 generation is detectible in the matrix and at the mitochondrial surface. The ROS generated and detected at locations immediately apposed to Ca 2þ -transport proteins, such as the IP3-receptor, may modify their function at a local level and contribute to the feed-forward cycle of Ca 2þ -dysregulation and subsequent cell death. 1107-Plat ER-Mitochondrial Ultrastructure in the Liver of Normal and Ethnaol-Fed Rats Gyorgy Csordas1, David Mankus2, Antony Anil Noronha1, Jan Hoek1, Carmen Mannella2, Gyorgy Hajnoczky1. 1Thomas Jefferson University, Philadelphia, PA, USA, 2Wadsworth Center, Albany, NY, USA. Chronic alcohol consumption causes severe pathology in liver, associated with altered metabolism and reduced ATP production. Liver mitochondria isolated from alcohol-fed animals show decreased capacity for electron transport and ATP synthesis, impaired calcium handling, and increased ROS. In parallel, the endoplasmic reticulum (ER) also shows increased ROS and a stress re- sponse. The close correlation between defects in mitochondria and ER is matched by recent evidence of the functional integration of cellular responses (including calcium signaling) involving these organelles. To evaluate the ultra- structural basis of alcohol-induced changes, we performed transmission elec- tron microscopy and electron tomography studies of rat liver. Hepatocytes showed a large number of mostly round mitochondrial cross sections, which oc- cupied ~20% of the cytoplasmic area. The cell cytoplasm in chronic ethanol- fed (9 months) condition exhibited reduced particle density, indicative of cell swelling, and contained large lipid vesicles. The mitochondria were generally intact but showed narrower intermembrane spacings within cristae and at the organelle periphery, consistent with low-scale matrix swelling. In both control and ethanol-fed conditions, mitochondria were typically surrounded by exten- sive ER, with cisternae sometimes sandwiched between neighboring mitochon- dria. In one case, ER was prominent at a site of mitochondrial fusion/fission. As previously reported, regions of close ER-mitochondrial association (20-60 nm) contained numerous ‘‘tethers’’ between outer mitochondrial membranes (OMM) and adjacent ER. While individual tethers were discernible, dense granular material (including ribosomes) within OMM-ER interfaces interfered with accurate quantitation of tethers. However, membrane spacings could be readily measured. It was found that the mitochondrial surface in close associ- ation with ER was significantly reduced in the chronic ethanol-fed condition as compared to control (17.5 5 7% vs. 39 5 2%). This might be expected to cause reduced calcium signaling between ER and mitochondria in liver after chronic alcohol ingestion. 1108-Plat Mitochondrial Nm23-H4/NDPK-D is Multifunctional: Intermembrane Cardiolipin Transfer Linked to Apoptosis Uwe Schlattner1, Malgorzata Tokarska-Schlattner1, Sacnicte Ramirez1, Yulia Y. Tyurina2, Andrew A. Amoscato2, Zhentai Huang2, Jianfei Jiang2, Mathieu Boissan3, Raquel F. Epand4, Dariush Mohammadsanyi2, Judith Klein-Seetharaman2, Richard M. Epand4, Marie-Lise Lacombe5, Valerian E. Kagan2. 1Joseph Fourier University and Inserm, Grenoble, France, 2University of Pittsburgh, Pittsburgh, PA, USA, 3Inserm, UMPC Université Paris 06 and Hôpital Tenon, Paris, France, 4McMaster University, Hamilton, ON, Canada, 5Inserm and UMPC Université Paris 06, Paris, France. Nm23-H4/NDPK-D forms symmetrical homohexameric complexes in the mitochondrial intermembrane space. The well established function of the en- zyme is phosphotransfer activity as a nucleoside diphosphate kinase (NDPK), using mitochondrial ATP to regenerate nucleoside triphosphates. Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardi- olipin, and in vivo to inner mitochondrial membranes. We show here that such protein/lipid complexes inhibit NDPK activity but are necessary for Nm23-H4 to function in selective intermembrane lipid transfer. Nm23-H4-deficient HeLa cells expressing either wild-type Nm23-H4 or a membrane-binding deficient mutant were analyzed by membrane fractionation and LC-ESI-MS. Data revealed that wild-type Nm24-H4 increased cardiolipin content in the outer mitochondrial membrane as compared to mutant enzyme. This correlated with higher susceptibility of wild-type enzyme expressing cells to rotenone-induced apoptosis as seen by increased annexin V binding, elevated caspase 3/7 activity and stimulated release of cytochrome c into the cytosol. Molecular modeling of Nm23-H4 binding with cardiolipin reveals potential intermembrane transfer mechanisms. We propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function, allowing either for phosphotransfer or for cardiolipin transfer, with a role in apoptotic signaling. Calcium-Induced ROS Generation during Ischemia Triggers mPTP-Dependent Cell Death during Reperfusion Dynamic Measurement of Ca2+-Induced Changes in Organelle-Specific Redox Microdomains ER-Mitochondrial Ultrastructure in the Liver of Normal and Ethnaol-Fed Rats Mitochondrial Nm23-H4/NDPK-D is Multifunctional: Intermembrane Cardiolipin Transfer Linked to Apoptosis