Hepatic ischemia/reperfusion (I/R) injury produces cell death in a variety of clinical settings. A mystery regarding I/R injury concerns changes that occur during ischemia that predispose to injury after reperfusion. In the previous period of support, we developed the hypothesis that ATP depletion during ischemia inhibits the H+-pumping vacuolar ATPase (V-ATPase) in lysosomes/endosomes to collapse their acidic pH gradient, which in turn causes release of lysosomal Fe2+ into the cytosol without lysosomal rupture. Mitochondria take up this Fe2+ via the mitochondrial Ca2+,Fe2+ uniporter (MCFU) to predispose to iron-catalyzed hydroxyl radical (OH??) formation (Fenton chemistry) with consequent activation of c-Jun N-terminal kinase (JNK), onset of the mitochondrial permeability transition (MPT) and cell death after reperfusion. We also hypothesize that sphingosine accumulates during ischemia, which inhibits cytochrome oxidase to promote generation the OH? precursors, H2O2 and superoxide (O2??), from the respiratory chain after reperfusion. Together these events synergize to cause lethal cell injury. Pursuing these hypotheses, in Specific Aim 1 we will determine whether DMT1 inhibitors prevent translocation of iron from lysosomes to mitochondria during ischemia and prevent oxidative stress, MPT onset and cell death after reperfusion. We have crossed phloxed DMT1 mice with Alb- Cre mice to create a hepatocyte specific DMT1 knockout. We expect that hepatocellular DMT1 deficiency will prevent lysosomal iron mobilization during ischemia and improve survival after IR. We will then assess whether DMT1 inhibitors and DMT1 deficiency decrease reperfusion injury in vivo utilizing intravital multiphoton microscopy. We will also clarify the specific individual roles of mitochondrial MCFU-mediated Fe2+ and Ca2+ uptake in cytoprotection.
Specific Aim 2 is based on our experiments showing that JNK inhibition and JNK2 deficiency decrease I/R injury. We will perform experiments with iron chelators, MCU inhibitors, JNK inhibition and knockouts and MPT inhibitors to test the hypothesis that JNK activation after IR is downstream of iron- dependent Fenton chemistry and upstream of MPT onset. In further in vivo experiments, we will perform phosphoproteomics to determine changes in the mitochondrial outer membrane phosphoproteome after IR that are sensitive to JNK inhibition/knockout.
Specific Aim 3 is based on preliminary studies showing that sphingosine accumulates during ischemia and inhibits mitochondrial cytochrome oxidase, we hypothesize that sphingosine accumulation during ischemia enhances formation of H2O2 and O2?? from the inhibited respiratory chain to fuel iron catalyzed OH??. We further hypothesize that inhibition of mitochondrial sphingosine kinase-2 (SK2) inhibition prevents sphingosine elimination by phosphorylation to sphingosine-1-phosphate. We will test elements of this hypothesis by measuring sphingosine metabolites, reactive oxygen species and mitochondrial function during I/R in relation to SK2 inhibition and SK2 deficiency. Overall, success of this project will lead to new mechanistic insights into I/R injury, novel drug targets and innovative translatable therapeutic strategies.

Public Health Relevance

Hepatic ischemia/reperfusion (I/R) injury produces cell death in a variety of clinical settings, including liver surgery, liver preservation for transplantation, veno-occlusive disease, hemorrhagic shock, heart failure and alcoholic liver disease, but a mystery remains concerning the changes that occur during ischemia that predispose to injury after reperfusion. This project addresses the novel hypothesis that ATP depletion during ischemia leads to iron translocation from lysosomes to mitochondria, which predisposes to sphingosine-enabled hydroxyl radical (OH?) formation with consequent activation of c-Jun N-terminal kinase (JNK), onset of the mitochondrial permeability transition (MPT) and cell death after reperfusion. Success of this project will lead to new mechanistic insights into I/R injury, novel drug targets and innovative translatable therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK073336-08
Application #
9330839
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Burgess-Beusse, Bonnie L
Project Start
2007-03-20
Project End
2018-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
8
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29403
Ramshesh, Venkat K; Lemasters, John J (2018) Imaging of Mitochondrial pH Using SNARF-1. Methods Mol Biol 1782:351-356
Zhong, Zhi; Lemasters, John J (2018) A Unifying Hypothesis Linking Hepatic Adaptations for Ethanol Metabolism to the Proinflammatory and Profibrotic Events of Alcoholic Liver Disease. Alcohol Clin Exp Res 42:2072-2089
DeHart, David N; Lemasters, John J; Maldonado, Eduardo N (2018) Erastin-Like Anti-Warburg Agents Prevent Mitochondrial Depolarization Induced by Free Tubulin and Decrease Lactate Formation in Cancer Cells. SLAS Discov 23:23-33
DeHart, David N; Fang, Diana; Heslop, Kareem et al. (2018) Opening of voltage dependent anion channels promotes reactive oxygen species generation, mitochondrial dysfunction and cell death in cancer cells. Biochem Pharmacol 148:155-162
Teplova, Vera V; Kruglov, Alexey G; Kovalyov, Leonid I et al. (2017) Glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria. J Bioenerg Biomembr 49:253-264
Baburina, Yulia; Odinokova, Irina; Azarashvili, Tamara et al. (2017) 2',3'-Cyclic nucleotide 3'-phosphodiesterase as a messenger of protection of the mitochondrial function during melatonin treatment in aging. Biochim Biophys Acta Biomembr 1859:94-103
Liu, Qinlong; Rehman, Hasibur; Krishnasamy, Yasodha et al. (2017) 8-pCPT-cGMP prevents mitochondrial depolarization and improves the outcome of steatotic partial liver transplantation. Int J Physiol Pathophysiol Pharmacol 9:69-83
Lemasters, John J (2017) Evolution of Voltage-Dependent Anion Channel Function: From Molecular Sieve to Governator to Actuator of Ferroptosis. Front Oncol 7:303
Lemasters, John J; Ramshesh, Venkat K; Lovelace, Gregory L et al. (2017) Compartmentation of Mitochondrial and Oxidative Metabolism in Growing Hair Follicles: A Ring of Fire. J Invest Dermatol 137:1434-1444
Hendriks, Giel; Derr, Remco S; Misovic, Branislav et al. (2016) The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding. Toxicol Sci 150:190-203

Showing the most recent 10 out of 81 publications