ABSTRACT
Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the alpha-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In '2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3'-azido-3'deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.
Subject(s)
Anti-HIV Agents/toxicity , DNA, Mitochondrial/metabolism , Myocardium/metabolism , Nucleoside-Phosphate Kinase/metabolism , Nucleosides/metabolism , Zalcitabine/analogs & derivatives , Zidovudine/toxicity , Animals , Anti-HIV Agents/metabolism , DNA Replication/drug effects , DNA, Mitochondrial/drug effects , Echocardiography , Emtricitabine/analogs & derivatives , Female , Humans , Hypertrophy, Left Ventricular/chemically induced , Hypertrophy, Left Ventricular/diagnostic imaging , Male , Mice , Mice, Transgenic , Mitochondria, Heart/drug effects , Mitochondria, Heart/ultrastructure , Myocardium/pathology , Myocardium/ultrastructure , Nucleoside-Phosphate Kinase/genetics , Phosphorylation , Ventricular Function, Left , Zalcitabine/metabolism , Zalcitabine/toxicity , Zidovudine/metabolismABSTRACT
Transgenic mice (TG) were used to define mitochondrial oxidative stress and cardiomyopathy (CM) induced by zidovudine (AZT), an antiretroviral used to treat HIV/AIDS. Genetically engineered mice either depleted or overexpressed mitochondrial superoxide dismutase (SOD2(+/-) KOs and SOD2-OX, respectively) or expressed mitochondrially targeted catalase (mCAT). TGs and wild-type (WT) littermates were treated (oral AZT, 35 days). Cardiac mitochondrial H(2)O(2), aconitase activity, histology and ultrastructure were analyzed. Left ventricle (LV) mass and LV end-diastolic dimension were determined echocardiographically. AZT induced cardiac oxidative stress and LV dysfunction in WTs. Cardiac mitochondrial H(2)O(2) increased and aconitase was inactivated in SOD2(+/-) KOs, and cardiac dysfunction was worsened by AZT. Conversely, the cardiac function in SOD2-OX and mCAT hearts was protected. In SOD2-OX and mCAT TG hearts, mitochondrial H(2)O(2), LV mass and LV cavity volume resembled corresponding values from vehicle-treated WTs. AZT worsens cardiac dysfunction and increases mitochondrial H(2)O(2) in SOD2(+/-) KO. Conversely, both SOD2-OX and mCAT TGs prevent or attenuate AZT-induced cardiac oxidative stress and LV dysfunction. As dysfunctional changes are ameliorated by decreasing and worsened by increasing H(2)O(2) abundance, oxidative stress from H(2)O(2) is crucial pathogenetically in AZT-induced mitochondrial CM.
Subject(s)
Anti-HIV Agents/toxicity , Cardiomyopathies/chemically induced , Cardiomyopathies/prevention & control , Catalase/metabolism , Mitochondria, Heart/drug effects , Mitochondria, Heart/enzymology , Superoxide Dismutase/metabolism , Zidovudine/toxicity , Aconitate Hydratase/metabolism , Animals , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Catalase/genetics , Female , Gene Expression , Hydrogen Peroxide/metabolism , Male , Mice , Mice, Congenic , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Electron, Transmission , Mitochondria, Heart/ultrastructure , Models, Cardiovascular , Myocardium/pathology , Oxidative Stress/drug effects , Phenotype , Superoxide Dismutase/deficiency , Superoxide Dismutase/geneticsABSTRACT
Tenofovir disoproxil fumarate (TDF) is an analog of adenosine monophosphate that inhibits HIV reverse transcriptase in HIV/AIDS. Despite its therapeutic success, renal tubular side effects are reported. The mechanisms and targets of tenofovir toxicity were determined using '2 x 2' factorial protocols, and HIV transgenic (TG) and wild-type (WT) littermate mice with or without TDF (5 weeks). A parallel study used didanosine (ddI) instead of TDF. At termination, heart, kidney, and liver samples were retrieved. Mitochondrial DNA (mtDNA) abundance, and histo- and ultrastructural pathology were analyzed. Laser-capture microdissection (LCM) was used to isolate renal proximal tubules for molecular analyses. Tenofovir increased mtDNA abundance in TG whole kidneys, but not in their hearts or livers. In contrast, ddI decreased mtDNA abundance in the livers of WTs and TGs, but had no effect on their hearts or kidneys. Histological analyses of kidneys showed no disruption of glomeruli or proximal tubules with TDF or ddI treatments. Ultrastructural changes in renal proximal tubules from TDF-treated TGs included an increased number and irregular shape of mitochondria with sparse fragmented cristae. LCM-captured renal proximal tubules from TGs showed decreased mtDNA abundance with tenofovir. The results indicate that tenofovir targets mitochondrial toxicity on the renal proximal tubule in an AIDS model.
Subject(s)
AIDS-Associated Nephropathy/chemically induced , Adenine/analogs & derivatives , Anti-HIV Agents/adverse effects , Kidney Tubules, Proximal/drug effects , Mitochondria/drug effects , Organophosphonates/adverse effects , AIDS-Associated Nephropathy/pathology , Adenine/adverse effects , Animals , DNA, Mitochondrial/metabolism , Didanosine/adverse effects , Female , HIV-1 , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/ultrastructure , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microdissection , Mitochondria/ultrastructure , Tenofovir , Urothelium/ultrastructureABSTRACT
Mitochondrial toxicity results from pyrimidine nucleoside reverse transcriptase inhibitors (NRTIs) for HIV/AIDS. In the heart, this can deplete mitochondrial (mt) DNA and cause cardiac dysfunction (eg, left ventricle hypertrophy, LVH). Four unique transgenic, cardiac-targeted overexpressors (TGs) were generated to determine their individual impact on native mitochondrial biogenesis and effects of NRTI administration on development of mitochondrial toxicity. TGs included cardiac-specific overexpression of native thymidine kinase 2 (TK2), two pathogenic TK2 mutants (H121N and I212N), and a mutant of mtDNA polymerase, pol-gamma (Y955C). Each was treated with antiretrovirals (AZT-HAART, 3 or 10 weeks, zidovudine (AZT) + lamivudine (3TC) + indinavir, or vehicle control). Parameters included left ventricle (LV) performance (echocardiography), LV mtDNA abundance (real-time PCR), and mitochondrial fine structure (electron microscopy, EM) as a function of duration of treatment and presence of TG. mtDNA abundance significantly decreased in Y955C TG, increased in TK2 native and I212N TGs, and was unchanged in H121N TGs at 10 weeks regardless of treatment. Y955C and I212N TGs exhibited LVH during growth irrespective of treatment. Y955C TGs exhibited cardiomyopathy (CM) at 3 and 10 weeks irrespective of treatment, whereas H121N and I212N TGs exhibited CM only after 10 weeks AZT-HAART. EM features were consistent with cardiac dysfunction. mtDNA abundance and cardiac functional changes were related to TG expression of mitochondrially related genes, mutations thereof, and NRTIs.
