Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals

Abstract

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

Figure 1

Real-time PCR mtDNA/nDNA ratios with NRTI treatment: TG and WT cohorts were treated with HAART [AZT, 3TC, and indinavir (IDV) for 35 days] or with vehicle control for 35 days. Tissue samples were analyzed using real-time PCR. A: TK1 + HAART resulted in no significant change from WT. B: An increase in mtDNA/nDNA ratio was found with TK2-HAART compared with WT.

Figure 2

Steady-state abundance of 17-kd subunit of complex I polypeptide by Western blot in TK1 (A) or TK2 (B) TGs with AZT-HAART: densitometric scans were performed using antibody to the 17-kd subunit of complex I and densities normalized to porin control on the same blot. All data are expressed as relative density. A small but significant increase (∼11%) in abundance of 17-kd subunit of complex 1 was found in untreated TK2 TGs compared with WT counterparts (P < 0.01). Treatment with combined antiretrovirals containing AZT reverted abundance of 17-kd subunit of complex I in TK2 TGs to that found in WT.

Figure 3

Electron photomicrographs of mitochondria from cardiac myocytes of TK2 TG and WT: TK2 overexpression in the heart increased mitochondrial abundance and cristae density (bottom left). The addition of HAART containing AZT resulted in some mitochondrial destruction with some loss of cristae (arrows), but abundant densely packed mitochondria remained. Scale bar = 1 μm. Original magnifications, ×26,000.

Figure 4

SDH histochemical staining in cardiac tissues from TK2 TGs and WTs: frozen cardiac tissues from representative TK2 TGs and WTs, with or without AZT-HAART (35 days), were sectioned and stained for SDH enzyme activity. Representative stained tissues for untreated WT (top left) or TK2 TGs (bottom left) and AZT-HAART-treated WT (top right) or TK2 TGs (bottom right) are shown. Increased dark blue staining is indicative of increased SDH enzyme activity. Darkest staining was found in TK2 + HAART sample (bottom right).

Figure 5

COX histochemical staining in cardiac tissues from TK2 TGs and WTs: frozen cardiac tissues from TK2 TGs and WTs, with or without AZT-HAART (35 days), were sectioned and stained for SDH enzyme activity. Representative stained tissues for untreated WT (top left) or TK2 TGs (bottom left) and AZT-HAART-treated WT (top right) or TK2 TGs (bottom right) are shown. Sites of COX enzyme activity have dark brown to red precipitation.

Figure 6

Quantitative analysis of echocardiographic images: LV mass was calculated in a blinded manner, code was broken, and data tabulated. A: TK1 TGs resulted in no change in LV mass from WT. B: TK2 TGs, both untreated and treated, had significant increases (∼25 to 50%) in LV mass from WT. Data were normalized to body weight (mg/g) and plotted as mean ± SEM.

Figure 7

Cardiac MRI of TK2 TGs and WT littermates: to evaluate LV cavity volume, TK2 TGs (n = 3; two treated, one untreated) and WT littermates (n = 3; two treated, one untreated) were evaluated via MRI at 16 to 20 weeks using a 4.7T Varian/INOVA (200/33) spectroscopy and imaging system. MRI images of hearts of TK2 TGs revealed increased end diastolic volume (LVEDV) (48.7 versus 61.2 μl, untreated versus treated, respectively) compared with LVEDV found in WT (∼34.5 μl for both treated and untreated). LVEDV of WT was essentially unchanged by administration of antiretroviral therapy.