Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
As a first step towards designing a cell line model to study mitochondrial disease we require tools to assess mitochondrial function and dysfunction in human cells. Here we use the fluorescent probe tetramethylrhodamine ethyl ester (TMRE) to assess membrane potential (DYm) in a hepatocyte-representative cell line, HepaRG. After investigating optimal conditions such as proliferative and differentiated HepaRG cell densities, concentration of TMRE and various concentrations of stressors required to inhibit the mitochondrial electron transport chain (ETC), a protocol has been established that will assist us in assessing the health of mitochondria when cells are exposed to xenobiotics or when a cell harbors a mitochondrial disease mutation. Using TMRE mitochondrial depolarization was measured when cells were exposed to 2.5 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore that dissipates DYm. TMRE was also used to measure HepaRG mitochondrial hyperpolarization upon exposure to 4 μM oligomycin, an inhibitor of the adenosine i triphosphate (ATP) synthase. Fluorescence data obtained from TMRE experiments were normalized to total cellular protein. Results from this work demonstrate that changes in DYm are adequately detected using our live-cell assay. The nucleoside reverse transcriptase inhibitors (NRTI) 2′-3′-dideoxycytidine (ddC, Zalcitabine) has been used in antiretroviral therapy regimes to treat human immunodeficiency virus the cause of acquired immune deficiency syndrome (AIDS). Proliferative HepaRG cells were grown in various concentrations of ddC for ten days. Cells treated with 0.25 – 12 μM ddC displayed a decrease in proliferation by day 8. When the DYm of ddC treated HepaRG cells was analyzed an ~2-fold decrease was observed relative to untreated cells. Analysis of HepaRG mitochondrial bioenergetics was performed using the Seahorse xFp extracellular flux analyzer. Spare respiratory capacity, proton leak and ATP-linked respiration bioenergetic parameters were investigated. Preliminary results suggest ddC treatment may cause a decrease in spare respiratory capacity. As ddC is an inhibitor of the replicative mitochondrial DNA (mtDNA) polymerase future studies analyzing mtDNA will be helpful to understand the links between mtDNA, bioenergetics, and DYm.
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