Integrated molecular, biochemical, imaging and pathologic approaches are used to define mechanisms of AIDS cardiomyopathy (CM) from nucleoside reverse transcriptase inhibitor (NRTI) therapy. Survival with AIDS improved with NRTIs (like AZT and D4T) but is limited by mitochondrial CM. The """"""""DMA pol y hypothesis"""""""" underscores NRTI mitochondrial import, phosphorylation by cellular kinases (to active moieties) and emphasizes inhibition of DNA pol y (the mitochondrial (mt-) DNA replicative enzyme) by NRTI triphosphates and resulting defective mtDNA replication. The """"""""mitochondrial dysfunction hypothesis"""""""" adds contributions of mtDNA mutations and oxidative stress (imbalance between reactive oxygen species and antioxidants). Experiments test this hypothesis: CM results from alterations in mitochondrial concentration of thymidine (dT) and NRTIs. Mitochondrial NRTI and dT concentrations are regulated by their import, phosphorylation and dephosphorylation. Together with DNApol y, these regulate mtDNA replication. Mitochondrial uptake and phosphorylation of NRTIs and NRTI effects on mtDNA replication are explored in vivo with myocardial-targeted gene expression (cardiac targeted TGs). The project uses TGs to evaluate cardiac mitochondrial dT and NRTI pools and import, phosphorylation, dephosphorylation, and mtDNA replication. The first TG overexpresses deoxynucleoside carrier (DNC) and imports phosphorylated dT (and AZT) into mitochondria. The mitochondrial equilibrative nucleoside transporter (ENT1) that imports NRTIs (like FIAU) also is expressed transgenically. Thymidine kinase 2 (TK2;phosphorylates pyrimidines intramitochondrially) is evaluated with """"""""dominant negative"""""""" mutant TGs. Mitochondrial NRTI dephosphorylation by deoxyribonucleotidase (mdN;balances TK2 activity) is analyzed by TG overexpression. Since intramitochondrial NRTI triphosphates inhibit DNA pol y, that enzyme's role in cardiac mtDNA replication is evaluated with TGs and NRTIs. Inhibited mtDNA replication, mtDNA depletion, and electron transport defects are expected phenotypes in experiments. HYPOTHESIS 1: DNC and/or ENT1 imports dT and dT-like NRTIs into mitochondria. Altered DNC or ENT1 activity alters dT and NRTI intramitochondrial pools for phosphorylation. Ultimately, intramitochondrial NRTI triphosphates inhibit mtDNA replication, decrease mtDNA, mtRNA and mitochondrial polypeptides, and""""""""result in CM. HYPOTHESIS 2: dT is phosphorylated intramitochondrially by TK2. Monophosphates are dephosphorylated by mdN. Increased activity of mdN (by overexpressing native mdN) or decreased activity of TK2 (by overexpressing mutant TK2 His121Asn, lle212Asn) disturbs mitochondrial dT (and NRTI) pools and cause CM. HYPOTHESIS 3: Mutant DNA pol y (Y955C) depletes mtDNA and causes mutations. NRTIs inhibit DNA pol y and deplete mtDNA. NRTI treatment of the DNA pol y Y955C TG yields additively or synergistically harmful effects. HYPOTHESIS 4: mtDNA depletion (from any above) causes or results from oxidative stress. Antioxidants ameliorate oxidative stress from NRTIs by decreasing oxidation of mtDNA.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Study Section
NeuroAIDS and other End-Organ Diseases Study Section (NAED)
Program Officer
Mcdonald, Cheryl
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Emory University
Schools of Medicine
United States
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Kushnir, Vitaly A; Lewis, William (2011) Human immunodeficiency virus/acquired immunodeficiency syndrome and infertility: emerging problems in the era of highly active antiretrovirals. Fertil Steril 96:546-53
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