Transgenic cardiac-targeted overexpression of human thymidylate kinase

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.

Figure 1. Generation and verification of TMPK transgenesis

A) Molecular map summarizes α-MyHC -TMPK construct with restriction sites and primer binding sites for positive identification of transgenes. Representative Dot blot (B) and agarose gel image of PCR amplification products (C) demonstrate selection of positive TGs from tail DNA of individual progeny from TG line(s) derived.

Figure 2. Cardiac mtDNA abundance in TMPK TGs and WTs following NRTI treatment

Total DNA were extracted from cardiac tissues isolated from TMPK TG and WT cohorts treated with zidovudine (AZT in CMC vehicle), racivir (RCV in saline vehicle) or vehicle alone for 35 days. mtDNA abundance was assessed using a ratio of mtDNA/nDNA as determined by real time PCR amplification. Cardiac mtDNA abundance was significantly decreased in TMPK TGs treated with AZT (A) or RCV (B) compared to WT littermates.

Figure 3. Electron photomicrographs of mitochondria from cardiac myocytes of TMPK TG and WT treated with AZT or RCV

Representative TEM profiles of cardiac tissues from “2×2” studies of TMPK TGs and WTs treated with AZT (A), RCV (B), and their respective vehicle controls are shown. Cardiac myocytes from TMPK TGs treated with AZT demonstrate disrupted sarcomeres and tubules with mitochondrial swelling and reduced cristae density (A, lower right panel) compared to vehicle-treated WTs. (B) Changes following treatment with RCV in TGs and WTs were unremarkable. (original magnification: X 22,300, Marker indicates 1μm).

Figure 4. Quantitative analysis of ECHO images

LV mass was determined from ECHO images captured just prior to termination. Data were normalized to body weight (mg/g) and plotted as mean±SEM. A) AZT treatment increased LV mass in WT and TGs, compared to vehicle-treated WTs (p<0.05). B) LV mass remained unchanged in TGs and WTs following RCV treatment (right graph).

Figure 5. Histological analysis of cardiac tissues from TMPK TGs and WTs treated with AZT or RCV

Parallel H&E –stained slides were made from gender-matched pairs of cardiac tissues after treatment (35 days). All tissues from TMPK TGs and WTs treated with AZT (A) or RCV (B) showed intact cardiomyocytes with comparable nuclei (original magnification x 40).

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