Azidothymidine-triphosphate impairs mitochondrial dynamics by disrupting the quality control system

Abstract

Highly active anti-retrovirus therapy (HAART) has been used to block the progression and symptoms of human immunodeficiency virus infection. Although it decreases morbidity and mortality, clinical use of HAART has also been linked to various adverse effects such as severe cardiomyopathy resulting from compromised mitochondrial functioning. However, the mechanistic basis for these effects remains unclear. Here, we demonstrate that a key component of HAART, 3ꞌ-azido-3ꞌ-deoxythymidine (AZT), particularly, its active metabolite AZT-triphosphate (AZT-TP), caused mitochondrial dysfunction, leading to induction of cell death in H9c2 cells derived from rat embryonic myoblasts, which serve as a model for cardiomyopathy. Specifically, treatment with 100µM AZT for 48h disrupted the mitochondrial tubular network via accumulation of AZT-TP. The mRNA expression of dynamin-related protein (Drp)1 and the Drp1 receptor mitochondrial fission factor (Mff) was upregulated whereas that of optic atrophy 1 (Opa1) was downregulated following AZT treatment. Increased mitochondrial translocation of Drp1, Mff upregulation, and decreased functional Opa1 expression induced by AZT impaired the balance of mitochondrial fission vs. fusion. These data demonstrate that AZT-TP causes cell death by altering mitochondrial dynamics.

Keywords: Cardiomyopathy; Metabolites; Mitochondrial dynamics; Mitochondrial function; Oxidative phosphorylation; Oxidative stress.

Fig. 1

TMPK expression in transduced H9c2 cells. A. TMPK expression in transduced H9c2 cells was examined by western blotting. GAPDH was used as a loading control. Lane 1, parental H9c2 cells; lane 2, cells transduced with LV/TMPK-WT; lane 3, cells transduced with LV/TMPK-mutant. B. Quantification of TMPK expression levels. Transduced cells showed higher TMPK expression than parental H9c2 cells. ***P < 0.001 and ****P < 0.0001 vs. parent. n.s., not significant vs. parent. Data represent mean ± SEM (n = 3). Statistical significance was evaluated by one-way analysis of variance with Bonferroni's multiple comparison post-hoc test.

Fig. 2

AZT metabolite levels in TMPK-expressing H9c2 cells. A. Representative chromatograms of AZT metabolites extracted from H9c2 cells treated with 500 µM AZT for 36 h and analyzed using reverse-phase HPLC. Retention times of AZT-MP (#), AZT-DP (##), and AZT-TP (###) are shown. B. Comparison of the ratio of intracellular AZT metabolite levels in TMPK-expressing H9c2 cells. Data represent mean ± SEM (n = 6). Statistical significance was evaluated with Student's t-test.

Fig. 3

Cell viability is decreased by active AZT metabolites. A. AZT-TP accumulation in mutant TMPK-expressing cells reduced cell viability in a concentration-dependent manner. Cells were cultured in the absence or presence of increasing concentrations of AZT for 48 h and viability was evaluated using the Cell Titer-Glo kit. Data represent mean ± SEM (n = 6). Statistical significance was evaluated by one-way analysis of variance with Bonferroni's multiple comparison post-hoc test. **P < 0.01, ****P < 0.0001 vs. vehicle. ○: WT TMPK; •: Mutant TMPK. B. Mutant TMPK with 100 µM AZT treatment for 48 h increases apoptosis. Data represent mean ± SEM (n = 3). Statistical significance was evaluated by one-way analysis of variance with Bonferroni's multiple comparison post-hoc test. ***P < 0.001 vs. mutant TMPK without AZT.

Fig. 4

Accumulation of AZT-TP but not AZT-MP disrupts the intracellular mitochondrial network. A. Mitochondrial network visualized by confocal microscopy after staining with 100 nM MitoTracker green. H9c2 cells expressing WT or mutant TMPK were treated with 100 µM AZT (+) or left untreated (−). Scale bar = 5 µm. B. Mitochondrial area per cell was decreased in mutant TMPK-expressing cells treated with AZT. *P < 0.05 vs. mutant TMPK without AZT; n.s., not significant vs. mutant TMPK without AZT. Data represent mean ± SEM (n = 13). Statistical significance was evaluated by one-way analysis of variance with Bonferroni's multiple comparison post-hoc test. n.s.; not significant vs. mutant TMPK without AZT.

Fig. 5

Changes in expression levels of mitochondrial fission-fusion related factors induced by AZT. H9c2 cells expressing mutant TMPK were treated with 100 µM AZT (+) or left untreated (−) and changes in the expression of Opa1, Drp1, and Mff were measured by quantitative real-time PCR. Data represent mean ± SEM (n = 3). Statistical significance was evaluated with Student's t-test.

Fig. 6

Expression of factors related to mitochondrial fission and fusion in H9c2 cells expressing mutant TMPK. A–C. H9c2 cells expressing mutant TMPK were treated with 100 µM AZT (+) or left untreated (−) and changes in (A) the ratio of Opa1-L to Opa1-S and the expression of (B) Drp1 and (C) Mff relative to voltage-dependent anion channel (VDAC) were evaluated by western blotting. D. Mitochondrial translocation of Drp1 was increased by AZT treatment. The ratio of p-Drp1 to Drp1 is shown. Data represent mean ± SEM (n = 3). Statistical significance was evaluated with Student's t-test. E. Drp1 translocation to mitochondria was increased by treatment with 100 µM AZT, as determined by immunohistochemistry. Data represent mean ± SEM (n = 50). Statistical significance was evaluated with Student's t-test.

Fig. 7

Evaluation of mitochondrial respiration. A. Representative OCR profiles of H9c2 cells treated with 100 µM AZT or left untreated. After measurement of basal OCR, cells were sequentially incubated with 1 µM oligomycin to measure proton leakage (a); 0.3 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone to measure maximum respiration capacity (b); and 0.1 µM rotenone and 0.1 µM antimycin A to terminate the reaction (c). ○: WT TMPK without AZT; •: WT TMPK with AZT; △: mutant TMPK without AZT; ▲: mutant TMPK with AZT. Data represent mean ± SEM (n = 5). B. OCR (basal level). C. OCR following oligomycin addition. D. OCR of maximum respiratory capacity. Statistical significance was evaluated by one-way analysis of variance with Bonferroni's multiple comparison post-hoc test. *P < 0.05 vs. mutant TMPK without AZT.

Fig. 8

AZT-TP accumulation causes mitochondrial dysfunction. A. AZT-TP accumulation severely decreases the mitochondrial membrane potential. H9c2 cells expressing WT or mutant TMPK were treated with 100 µM AZT (+) or left untreated (−) and mitochondrial membrane potential was monitored using the mitochondrial membrane potential sensor JC-1. The ratio of red to green JC-1 fluorescence intensity is shown. Data represent mean ± SEM (n = 3). *P < 0.05 and ***P < 0.0001 vs. mutant TMPK without AZT (−). B. AZT-TP accumulation increases ROS production. H9c2 cells expressing WT or mutant TMPK were treated with 100 µM AZT (+) or left untreated (−) and ROS levels were measured with the superoxide anion-specific fluorescent probe DHE; fluorescence intensity data are shown as mean ± SEM (n = 3). ***P < 0.0001 vs. mutant TMPK without AZT (−). C. AZT-TP accumulation causes mPTP opening. H9c2 cells expressing WT or mutant TMPK were treated with 100 µM AZT (+) or left untreated (−) and mPTP opening was measured with the calcein-cobalt method and is shown as a relative percentage (means ± SEM) of the control (n = 3). **P < 0.001 and ***P < 0.0001 vs. mutant TMPK without AZT (−). Statistical significance was evaluated by one-way analysis of variance with Bonferroni's multiple comparison post-hoc test.

Fig. 9

Ratio of mtDNA/nDNA is unaffected by AZT treatment. H9c2 cells expressing WT or mutant TMPK were treated with 100 µM AZT (+) or left untreated (−). A. Ratio of cytochrome oxidase (CO)1 to polymerase gamma 2 accessory subunit (PG2AS). B. Ratio of adenine dinucleotide hydride dehydrogenase (ND)1 to PG2AS. Data represent mean ± SEM (n = 3). Statistical significance was evaluated by one-way analysis of variance with Bonferroni's multiple comparison post-hoc test.